*Article* **Assessment of Physicochemical and In Vivo Biological Properties of Polymeric Nanocapsules Based on Chitosan and Poly(***N***-vinyl pyrrolidone-***alt***-itaconic anhydride)**

**Kheira Zanoune Dellali 1 , Mohammed Dellali 1 , Delia Mihaela Ra¸tă 2, \* , Anca Niculina Cadinoiu 2 , Leonard Ionut Atanase 2, \* , Marcel Popa 2,3, \* , Mihaela-Claudia Spataru <sup>4</sup> and Carmen Solcan 4**


**Abstract:** Drug delivery is an important field of nanomedicine, and its aim is to deliver specific active substances to a precise site of action in order to produce a desired pharmacological effect. In the present study nanocapsules were obtained by a process of interfacial condensation between chitosan (dissolved in the aqueous phase) and poly(*N*-vinyl pyrrolidone-*alt*-itaconic anhydride), a highly reactive copolymer capable of easily opening the anhydride ring under the action of amine groups of chitosan. The formed amide bonds led to the formation of a hydrogel membrane. The morphology of the obtained nanocapsules, their behavior in aqueous solution of physiological pH, and their ability to encapsulate and release a model drug can be modulated by the parameters of the synthesis process, such as the molar ratio between functional groups of polymers and the ratio of the phases in which the polymers are solubilized. Although a priori both polymers are biocompatible, this paper reports the results of a very detailed in vivo study conducted on experimental animals which have received the obtained nanocapsules by three administration routes—intraperitoneal, subcutaneous, and oral. The organs taken from the animals' kidney, liver, spleen, and lung and analyzed histologically demonstrated the ability of nanocapsules to stimulate the monocytic macrophage system without producing inflammatory changes. Moreover, their in vivo behavior has been shown to depend not only on the route of administration but also on the interaction with the cells of the organs with which they come into contact. The results clearly argue the biocompatibility of nanocapsules and hence the possibility of their safe use in biomedical applications.

**Keywords:** nanoparticles; natural and synthetic polymers; drug delivery systems; biocompatibility; in vivo tests

#### **1. Introduction**

Nanotechnology has gained considerable attention during last decades, and the development of different types of nanoparticles for biomedical applications is a growing field of research. Over the past twenty years, a significant number of nanoparticulate systems, composed of different materials including lipids, polymers, and inorganic materials, have been proposed in the biomedical field [1–3]. These nanocarriers, usually ranging from 1 to 1000 nm and suitable for the delivery of drugs, hormones, genes, nucleic acids, or imaging agents, have been designed in order to obtain improved specificity, drug targeting, and delivery efficiency, thus reaching a maximal therapeutic effect with minimal side effects [4].

**Citation:** Dellali, K.Z.; Dellali, M.; Ra¸t ˘a, D.M.; Cadinoiu, A.N.; Atanase, L.I.; Popa, M.; Spataru, M.-C.; Solcan, C. Assessment of Physicochemical and In Vivo Biological Properties of Polymeric Nanocapsules Based on Chitosan and Poly(*N*-vinyl pyrrolidone-*alt*-itaconic anhydride). *Polymers* **2022**, *14*, 1811. https://doi.org/10.3390/ polym14091811

Academic Editors: Agnieszka Tercjak and Stefano Leporatti

Received: 17 February 2022 Accepted: 19 April 2022 Published: 28 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Drug delivery is an important field of nanomedicine, and its aim is to deliver specific active substances to a precise site of action in order to produce a desired pharmacological effect. The development of a drug delivery system is influenced not only by the target site but also by the route of administration and the nature of the nanocarriers [5].

Polymer-based vehicles constitute the main branch of drug delivery systems and can be used for both diagnostic and therapeutic purposes. Polymer carriers with a spherical shape and smooth texture are considered ideal for delivering chemotherapeutic agents in order to easily transport them through the vascular system [6]. The most-investigated polymer carriers are micelles, nanospheres, nanocapsules, dendrimers, and polymersomes [7–10]. Generally, all these carriers can increase the local concentration of loaded active substances and improve their delivery, especially when they are poorly water soluble or when their bioavailability is low.

At the present, the majority of studies regarding polymeric drug delivery systems are focused on optimizing the nanocarrier physicochemical parameters, such as size, physical stability, and drug loading efficacy, but also on carrying on preliminary in vitro cytotoxicity tests in order to prove the effectiveness of the obtained formulations [11]. However, in vivo tests, which are useful in the investigation of the biological effects of these polymeric nanomaterials, are often not taken into account as they can be difficult to carry out. Despite the remarkable rapidity of development of nanomedicine, relatively little is known about the interaction at the nanoscale of polymeric carriers with living systems. The behavior of nanocarriers in the body depends not only on the administration route but also on their interactions with the cells with which they come into contact.

Thus, in the present study, a series of original polymeric nanocapsules (NCs) based on chitosan (CS) and poly(*N*-vinyl pyrrolidone-*alt*-itaconic anhydride) (NVPAI), were firstly investigated from a physicochemical point of view, and then their biological features were determined by in vivo testing in order to demonstrate their effectiveness as safe drug delivery systems. At this point it is worth mentioning that the nanocapsules (NCs), due to their morphology, have some practical advantages with respect to other nanocarriers, as they can be characterized by increased drug encapsulation efficiency and thus by an enhanced therapeutic effect. According to its polarity, the active substance is incorporated in the core, which then acts as a reservoir, or possibly adsorbed or covalently attached to the polymeric shell [12].

These hollow NCs were obtained by interfacial condensation method in absence of any kind of toxic crosslinking agents and in normal conditions (temperature and pressure). The polymer shell was formed by amide bridges at the contact between the NH<sup>2</sup> groups of CS and highly reactive anhydride cycles of poly(NVPAI). It has to be mentioned that a similar type of NCs was already prepared and characterized by our research group [13], but the novelty of the present system is that the exterior layer of the polymeric shell of the NCs is formed by the synthetic NVPAI copolymer.

CS was used in this study as it is one of the biopolymers that can form nanoparticles with unique properties and therefore is currently receiving great interest for drug delivery and tissue engineering applications in the medical and pharmaceutical field due to its interesting features, such as biocompatibility, biodegradability, and non-toxicity [14]. Compared with natural polymers, synthetic polymers such as NVPAI have high purity and good reproducibility and can control the release time of loaded active substances [15].

The obtained NCs have been analyzed structurally by Fourier transform–infrared spectroscopy (FT-IR), morphologically by TEM, and gravimetrically by thermo-gravimetric analysis (TGA). Moreover, the resulting NCs were characterized in terms of particle sizes, zeta potential, drug encapsulation efficiency, and in vitro drug release kinetics by using a hydrophilic model drug, 5-fluorouracil (5-FU). Furthermore, in vivo tests were performed on adult albino BALB/c line mice using different concentrations of NC suspensions and three types of administration routes, such as intraperitoneally, subcutaneously, and per o.s. for 21 days.

#### **2. Experimental Part (Materials and Methods)**

#### *2.1. Materials*

Chitosan (CS) (low molecular weight, degree of deacetylation 91%), acetone, dimethyl sulfoxide (DMSO), hexane, surfactants (Tween 80, Span 80), and drug (5-Fluorouracil), were purchased from Sigma Aldrich (St. Louis, MO, USA). Poly(N-vinylpyrrolidone-alt-itaconic anhydride) (NVPAI) is an alternant copolymer synthesized in laboratory by a radical copolymerization method [13]. Also, phosphate-buffered solution (PBS) with pH = 7.4 and double-distilled water was prepared in our laboratory.

#### *2.2. Preparation Method of Nanocapsules*

CS/poly(NVPAI)-based nanocapsules (NCs) were obtained by the interfacial condensation method. Initially, the polymer solutions were prepared in different phases. The aqueous phase was obtained by dissolving a specific amount of CS (Table 1) in 20 mL of 2% acetic acid solution (0.4 mL acetic acid was added to 20 mL distilled water) at a temperature of 65 ◦C under magnetic stirring. The solution was filtered before use and brought to room temperature. Then, an appropriate quantity of non-ionic surfactant Tween 80 (2% *w*/*v*) was added in the polymer solution and well homogenized. Separately, the organic phase was prepared by dissolving exactly 500 mg of poly(NVPAI) in 15 mL of DMSO under magnetic stirring. After complete dissolution of the copolymer, a specific volume of acetone was added, according to Table 1, under continuous stirring followed by the addition of the hydrophobic surfactant Span 80 (2% *w*/*v*). After this dissolution step, the aqueous solution of CS was slowly added drop-wise into the organic solution of poly(NVPAI) under vigorous magnetic stirring at room temperature. After 2 h, the formed NCs were separated from supernatant by centrifugation for 20 min at 7500 rpm. NCs were then purified by successive washes with distilled water (7 times) and acetone (5 times). After the last wash with acetone, the product was washed twice with hexane. Finally, the obtained product was dried from hexane at room temperature to constant weight.


**Table 1.** Experimental parameters used for the preparation of NCs.

The variables taken into account in this study were the molar ratio between the functional groups involved in the condensation reaction, respectively NH2/anhydride cycles, expressed in the Table 1 as the ratio between the two polymers [CS/poly (NVPAI)], and the volume ratio between the aqueous and organic phases.

The yield of the NCs (Table 2) was calculated according to the following equation:

$$\text{NCs yield} \left( \% \right) = \frac{amount \ of \text{ recovered} \; nanocapsules}{total \; amount \ of \text{ polygons} \; used} \, \* \; 100 \tag{1}$$


**Table 2.** Yield data for the obtained NCs.

#### *2.3. Characterization Methods*

#### 2.3.1. Structural Characterization

The structural characterization of NCs was accomplished spectrally by Fourier transform infrared spectroscopy (Schimadzu Corporation, Kyoto, Japan) (FTIR) to confirm the formation of new amide groups. The structural characterization was performed with a IRSpirit FTIR Spectrometer spectrometer. All samples were prepared as KBr pellets and scanned over the wave number range of 400–4000 cm−<sup>1</sup> at a resolution of 4.0 cm−<sup>1</sup> . The relevant bands in the absorption spectrum have been attributed to corresponding functional groups.

#### 2.3.2. Size, Morphology and Zeta Potential of NCs

Transmission electron microscopy (TEM) was used to determine the size, shape, and surface morphology of NCs. The samples for transmission electron microscopy (TEM) were prepared by slow evaporation of a suspension in acetone on a formvar-coated copper grid. The samples were analyzed with a Philips CM100 microscope equipped with an Olympus camera and transferred to a computer equipped with the Megaview system.

The mean diameter of NCs in dispersion and the size distribution were determined in triplicate at 25 ◦C at a suspension concentration of 1% (*w*/*v*) by dynamic light scattering (DLS) (Zeta Nanosizer Malvern). Anhydrous acetone was used as a dispersant to evaluate the average diameter of unswollen NCs. The particles' diameter in physiological saline solution was also evaluated, this medium being similar to the in vivo medium. This evaluation was performed as soon as possible after NCs came into contact with the aqueous environment. The zeta potential of NCs was determined by electrophoresis in phosphate buffer solution (PBS; pH = 7.4).

#### 2.3.3. Thermal Properties

Thermogravimetric analyses (TGA) have allowed the determination of sample weight loss as a function of temperature. These analyses were accomplished with a TA Instrument Q600 analyzer in air atmosphere (100 mL/min) with a heating rate of 10 ◦C/min, in a temperature range from the room temperature to 700 ◦C. The nanocapsules samples with a weight between 8–10 mg were heated in a platinum crucible. The operation parameters were kept constant for all the tested samples. The thermal analysis results were processed with the Universal Analysis (V 2.0) software (TA instruments, New Castle, DE, USA).

#### 2.3.4. Swelling Behaviour in Aqueous Solutions

In order to predict and understand the behaviour of NCs during the encapsulating and release process, and therefore to assess their behaviour as potential drug carriers, swelling studies were performed by gravimetric method. The swelling degree of the NCs samples was analyzed in slightly alkaline aqueous medium, pH = 7.4, that simulates physiological conditions. A specific amount (0.03 g) of dried NCs were weighted and immersed in Eppendorf's tube containing PBS. The obtained suspension was maintained at 37 ± 0.5 ◦C under magnetic stirring at 120 rpm. At pre-set times, the suspension was centrifuged, the supernatant was removed, and the swollen sample was weighed. All experiments were performed in triplicate. To ensure the swelling until equilibrium, the samples were allowed to swell for 24 h. The percentage of swelling ratio (*Q*%) was determined with Equation (2)

$$Q(\%) = \frac{W - W\_0}{W\_0} \ast 100\tag{2}$$

where *W* is the weight of swollen sample (mg) and *W*<sup>0</sup> is the initial weight of dry sample (mg).

In parallel with the gravimetric method, the change in the size of the NCs on swelling was also evaluated.

#### 2.3.5. Drug Encapsulating Studies

The drug encapsulating process was carried out through diffusional mechanism. In this study, 5-Flourouracil (5-FU) was used as the model drug. Briefly, 20 mg NCs were dispersed in 1.5 mL aqueous drug solution with a concentration of 10 mg 5-FU/mL in ultrapure water. The suspension was maintained under magnetic stirring (120 rpm) and temperature (37 ◦C) for 24 h. The drug-loaded NCs were separated from supernatant by ultracentrifugation at 8000 rpm for 10 min. The drug-loaded NCs were lyophilized and stored as powder for further analyses. The amount of 5-FU encapsulated into NCs was calculated by the difference between the initial amount of 5-FU in solution and the amount of 5-FU from supernatant using a UV Spectrometer (Nanodrop One, Thermo Scientific, Waltham, MA, USA) at 266 nm [13,16]. The encapsulation efficiency (*Eef*%) of 5-FU into NCs was calculated as follows:

$$
\mu m\_l = m\_i - m\_s \tag{3}
$$

$$E\_{ef}\% = \frac{m\_i - m\_s}{m\_i} \times 100\tag{4}$$

where *ml*—the amount of encapsulated 5-FU (mg); *mi*—the initial amount of 5-FU (mg); *ms*—the amount of 5-FU found in supernatant (mg).

The obtained drug-loaded NCs are designated as follows: CN-1-5FU, CN-2-5FU, CN-3-5FU, CN-4-5FU, CN-5-5FU, CN-6-5FU, and CN-7-5FU.

#### 2.3.6. In Vitro Drug Release

The in vitro drug release studies were realized by the dialysis method. Each sample of 5-FU-encapsulated NCs was introduced into a dialysis membrane and, after that, was individually immersed in flasks with 13 mL PBS at pH = 7.4, a value which is similar to blood. This system was maintained at 37 ± 0.5 ◦C under continuous stirring at 120 rpm for all of the release period. At regular time intervals, 1 mL of solution was taken and replaced with fresh PBS. The 5-FU released and present in the medium was spectrophotometrically determined at 266 nm wavelength, using a Nanodrop One (Thermo Scientific). The release efficiency of 5-FU (*Ref*%) was calculated using Equation (5):

$$\text{Ref}(\%) = \frac{m\_r}{m\_l} \times 100\tag{5}$$

where *mr*—the amount of drug released from NCs (mg); *ml*—the amount of the drug encapsulated into the NCs (mg).

#### 2.3.7. In Vivo Testing

The study was conducted on 42 adult albino BALB/c line mice, aged approximately 4 months, reared under conventional laboratory conditions (20–23 ◦C, 55% UR), fed standardized pelleted feed, fruits and vegetables, and water at discretion. Their bodyweight was monitored in the first, tenth, and last day of the experiment, and it was constant within the experimental error limits (Figure S1). Mice undergoing the experiment (equally female and male) were divided into 7 groups: one control and 6 experimental groups of 6 mice. To each group, CN-4 and CN-6 suspensions were administered daily by three different routes, such as intraperitoneally (0.01 mL and 0.02 mL), subcutaneously (0.1 and 0.2 mL), and per o.s. (0.2 and 0.3 mL) for 21 days. Suspensions of CN-4 and CN-6 were obtained by adding 5 mL of saline solution over 6.25 mg powder, the suspension being prepared approximately 2 h before use. The health status of the mice and body weight dynamics were followed and 2 days after the completion of the experiment the mice were euthanized by cervical

dislocation and probes were harvested from internal organs (kidney, liver, spleen, and lung) and processed for histological examination.

#### 2.3.8. Histological and Immunohistological Analysis

Organs samples were fixed in 10% formalin solution for 24 h. Approximately 0.5 cmthick slices were dehydrated with a decreasing concentration of ethanol solution, then clarified in xylene and embedded in paraffin. After being cut with the microtometer, 10 microscope slides from each paraffin block were selected, specifically stained, and read on the Olympus CX41 microscope. They were initially stained with hematoxylin eosin (HE) then IHC using 4 antibodies: Cd147, p65, alpha SMA, HMC II, and Cox-2. Anti p65 (Nuclear factor-kB p65) antibodies, AA 143–158, antibodies, CD147 (ab188190), α-SMA (anti-alpha smooth muscle actin antibody), MA5-11547 (14A-asm-1), MHC II (Dako M0746), and Cox-2 (ab16701 SP-21), were used to perform immunohistochemical staining. After sections were deparaffinized in Xylen, hydrated in ethanol, and microwaved for 10 min at 95 ◦C in 10 mmol citrate acid buffer pH6, they were cooled for 20 min, then washed twice in PBS for 5 min. Slices were treated with 3% hydrogen peroxide and rinsed with PBS, after which they were incubated overnight at 4 ◦C in a humid atmosphere with primary antibodies in dilutions of 1:100 CD147, MHC II, Cox-2 and 1:500 p65, α-SMA. The following day, slides were washed 3 times in PBS for 5 min, being incubated with secondary antibodies. For CD147, MHC II, and Cox-2 activity of bone cells, goat anti-rabbit IgG secondary antibody was used, and for p65 and α-SMA, goat anti-mouse IgG secondary antibody was chosen. Microscope slides were developed in 3,3′ -diaminobenzidine (DAB) and finally counterstained with hematoxylin. Images were interpreted using ImageJ IHC profile software.

DAB IHC profile scores were negative (−), low positive (±), positive (+), and over positive (++). Scoring of HE histological lesions was done by assessing changes and scoring as follows: no change (−), minor (+), medium (++), and major (+++).

#### *2.4. Statistical Analysis*

The statistical significance of cytotoxic activity was analyzed by Student's *t*-test. The values are expressed as mean ± SE of three parallel measurements, *p* < 0.05 being considered significant.

#### **3. Results and Discussion**

The objective of the present study was to assess the physicochemical and biological properties of a series of NCs based on CS and poly(NVPAI) which can be further used as drug delivery systems for the controlled and sustained release of different types of drugs.

A first result is that the yield of obtaining NCs increases with increasing the amount of CS in their composition (respectively of the CS/poly(NVPAI) molar ratio), as can be noticed in Table 2.

The increase in the initial amount of chitosan leads to the increase of the bonds formed between the poly(NVPAI)- and CS-reactive groups because a higher number of these reactive groups are available and can be involved in the interfacial polycondensation reaction. Consequently, a smaller amount of non-crosslinked polymer chains remains in the system and can be eliminated during the purification and washing process of the nanocapsules. In addition, enhancement of the aqueous phase/organic phase ratio, at the same CS/poly(NVPAI) ratio, has led to a visible increase in the nanocapsules final yield [17].

Different analysis techniques were used, and the obtained results are presented in the following.

#### *3.1. FTIR Spectroscopy*

The FT-IR spectra of CS, poly (NVPAI), and NCs presented in Figure 1 confirmed the reaction between the anhydride groups of the copolymer and chitosan amine groups. Absorption bands at approximately 1778 cm−<sup>1</sup> and 1858 cm−<sup>1</sup> from the copolymer spectrum correspond to the stretching vibration of the –C=O anhydride groups. Another important peak is located at approximately 989 cm−<sup>1</sup> and can be assigned to the C–O–C bond in the anhydride group [13]. −

− −

**Figure 1.** FTIR spectra of CS, NVPAI, CN-4 and CN-6 samples.

− − CS sample presents a peak at 1652 cm−<sup>1</sup> that corresponds to carbonyl groups (C=O) and another peak at 1601 cm−<sup>1</sup> attributed to free amino groups (–NH2).

− − − − − The FT-IR spectra of NCs samples evidenced the disappearance of characteristic peaks for the anhydride groups and the appearance of the absorption bands at 1647 cm−<sup>1</sup> and 1658 cm−<sup>1</sup> corresponding to a carbonyl bond of the newly formed amide groups. The appearance of absorption bands at 1715 cm−<sup>1</sup> (for sample CN-6) and at 1721 cm−<sup>1</sup> (for sample CN-4) that are attributed to the –C=O of the carboxylic groups reveals that some of the anhydride groups have hydrolyzed. Finally, the disappearance of absorption bands at 989 cm−<sup>1</sup> evidenced that all anhydride groups participated in the amidation or hydrolysis reaction.

Figure 2 shows the FTIR spectra for simple 5-FU, CN-6 without drug and drug-loaded NCs (CN-4-5FU and CN-6-5FU). The peaks characteristic of 5-FU spectrum (1429.92 cm−<sup>1</sup> , 1246.89 cm−<sup>1</sup> , 812.19 cm−<sup>1</sup> , 755.00 cm−<sup>1</sup> , and 640.6 cm−<sup>1</sup> ) are also found in the NC samples loaded with the model drug. In addition, these peaks are not visible in the spectrum of samples without drug (Figures 1 and 2).

− − − − −

**Figure 2.** FTIR spectra of simple 5-FU, CN-6 without drug, and drug-loaded NCs (CN-4-5FU and CN-6-5FU).

#### *3.2. Size, Morphology and Zeta Potential of NCs*

The mean diameters for NCs in acetone varied between 107 and 250 nm (Figure 3 and Table 2). Size distribution curves evidenced the presence of two populations of nanocapsules in the case of CN-1, CN-2, and CN-3 samples. CN-4, CN-5, CN-6, and CN-7 samples presented a monomodal distribution. From the obtained results, it can be noticed that the NCs' diameter increases with the increasing of the quantity of CS. The explanation of this behavior is based on the fact that, by increasing the amount of CS, increasing amounts of polysaccharide are involved in the formation of the NCs' membrane, which becomes thicker, thus contributing to the increase of the diameter.

− − tt

− − − − − − −

**Figure 3.** Size distribution curves of NCs in acetone at room temperature.

Increasing the volume of the organic phase, the number of macromolecules of copolymer that come into contact with a drop of the aqueous solution of CS is decreased gradually due to the dilution of the copolymer solution, and therefore the diameter of NCs decreases.

The NCs swell very quickly in aqueous environments. After 5 min in the physiological saline solution, diameters between 1089 and 2256 nm were recorded. The amount of CS and the volume of the organic phase had the same influence on the NCs size, even in an aqueous environment.

The zeta potential values evidenced the stability of the aqueous dispersion of NCs in a medium with slightly alkaline pH. In Table 3 the zeta potential values are presented which varied between −8 mV and −23.21 mV. Negative charge of NCs can be attributed to the presence of carboxylic groups formed as a result of the interfacial condensation reaction by amidation of the anhydride cycle in poly (NVPAI) or by their hydrolysis. By condensation, a carboxylic group also appears in the reaction of an amine cleavage with the copolymer. By hydrolysis, each anhydride cycle generates two carboxylic groups. This explains why by increasing the amount of CS, the zeta potential is reduced: the explanation lies in the overall formation of fewer carboxylic groups. From Table 3 it can be seen that, in the case of NCs without 5-FU, the increasing of the CS amount in the system leads to a decrease in the zeta potential values. On the other hand, NCs loaded with 5-FU show an increase in the zeta potential value as the amount of CS in the composition of the NCs increases.


**Table 3.** Diameter in volume, polydispersity index (PDI), and Zeta potential values of NCs samples.

From the Figure 4 it appears that the NCs have a spherical morphology and a moderate polydispersity, but that their size is smaller than that in acetone obtained by DLS. This can probably be explained by the different principle of operation of the two techniques (in liquid media and in dry state). The solvent used for DLS, acetone, may diffuse to some extent inside the NCs causing a slight increase in its volume.

**Figure 4.** TEM images of CN-1, CN-2, CN-4 and CN-6 samples.

#### *3.3. Thermal Behaviour*

The evaluation of the temperature behavior of the NCs was performed, on one hand, in order to have additional evidence that their membrane is made of both polymers, and on the other hand, to determine whether it is possible to heat sterilize them before administration without suffering changes caused by possible thermal degradation. In Figure 5 are presented the TGA chromatograms of different NC samples.

− − − − − − −

**Figure 5.** TGA chromatograms of NCs samples.

From the TGA results provided in Figure 5, it is obvious that the obtained NCs are stable until 400 ◦C, when a second degradation step occurs. This is a proof that these NCs are stable during the sterilization step. Moreover, analysis of the TGA chromatograms

showed that the composition of the NCs shell does not qualitatively influence its thermal degradation behavior. It is found that indeed the CN-6 sample (with a higher yield, previously explained by the higher amount of reacted copolymer) has a slightly higher calcination residue content compared to the CN-2 sample.

#### *3.4. Swelling Degree of NCs*

The swelling behaviour of the obtained NCs was investigated in slightly alkaline medium (pH 7.4). This medium was chosen as these drug delivery systems are intended to be used in biological fluids, and the obtained results are illustrated in Figure 6.

**Figure 6.** The swelling kinetics curves of the NCs in alkaline conditions (pH = 7.4) for samples (**a**) CN-1, CN-2, CN-3, and CN-4; (**b**) CN-2, CN-5, CN-6, and CN-7.

It is evident that all the NCs presented a high swelling degree which varies from 1476% to 1851%. The size, the composition, and the preparation parameters of NCs have an important influence on the swelling properties. In Figure 6a it can be observed that the swelling degree varied between 1851% and 1476% and decreased with the increase of the molar ratio of CS/NVPAI. The highest swelling degree was obtained for the CN-1 sample which is characterized by the smallest CS amount. The swelling of the NCs is caused by the water penetration within the empty core until complete filling as well as by the swelling of the polymer membrane, which has a hydrogel character [17].

The increase in the number of moles of -NH<sup>2</sup> groups from CS has as a consequence the increasing of the crosslinking density of the network of which the capsule membrane is formed, which reduces the diffusion of the water inside the capsule but also the amount of water that swells the membrane. In the other hand, it can be observed that the swelling degree of the NCs increases with the increase of the volume organic phase (Figure 6b) for an identical CS/poly(NVPAI) molar ratio of 0.3. This behaviour is explained as follows: as the volume of the organic phase increases (dilution of the copolymer solution), the number of macromolecules of poly(NVPAI) which come into contact with CS at the interface of the aqueous solution with organic phase is decreased and becomes smaller and smaller, so the crosslinking density of the NCs membrane will decrease, and therefore, the swelling rate will increase [18].

Since NCs can be used in physiological saline solutions for medical applications, it has also been necessary to evaluate their size in this environment (Figure 7). For the CN-4 and CN-6 samples, which were used for in vivo tests, the influence of the aqueous medium on the size over time was also evaluated (Figure 8).

After 24 h the diameter of the NCs had values between 5700 and 10,300 nm. Being capsules, it is easy to understand why they grow so much in size in the aqueous environment. Aqueous solution penetrates very easily inside the capsule and the network that creates the membrane of the capsule can relax freely.

Figure 8 shows that the diameter of the two samples increases quite rapidly after they are introduced into the saline solution. After only 2 min they go from nanometers to micrometers, and after half an hour they almost reach equilibrium.

**Figure 7.** Evaluation of the average diameter of the NCs after 2 min and 24 h in physiological saline solutions.

**Figure 8.** The influence of the aqueous environment on the size of CN-4 and CN-6 samples over time.

#### *3.5. Encapsulation Efficiency of a Model Drug*

This type of NC was developed in order to be used as efficient drug delivery systems for hydrophilic drugs. In this study, 5-FU was used as a model drug. The efficiency of encapsulating 5-FU into NCs and the amount of encapsulated drug (5-FU g/g NCs) are presented in Table 4.


**Table 4.** Drug encapsulation efficiency values.

#### *3.6. Drug Release Kinetics*

The release studies of the model drug, 5-FU, were performed in slightly alkaline medium (pH = 7.4) and the results are reported in Figure 9.

**Figure 9.** In vitro release kinetics of 5-FU from NCs in phosphate buffer solution (pH 7.4), with a zoom insert of the release kinetics between 0 and 30 min, for samples (**a**) CN-2, CN-3, and CN-4; (**b**) CN-5, CNM-6, and CN-7.

The results showed that 5-FU release from NCs was between 51% and 60%, and simple 5-FU was 100% released within 270 min of the start of the experiment. From Figure 9, it appears also that in the case of NCs loaded with 5-FU, a faster release was observed in the first hour which is due to the release of the drug adsorbed at the surface of NCs followed by a slow release of the encapsulated drug until the equilibrium was reached. Although the differences in release kinetics between the obtained NCs samples are not noticeable, there is still an influence on the amount of CS. If the CS amount increases, the reticulation density increases also, leading thus to a decrease of the drug release rate through the NCs shell. These results are in full accordance with the evolution of the previously discussed swelling process. These tests indicate that the obtained NCs can be used for the controlled and sustained release of hydrophilic drugs.

#### *3.7. In Vivo Testing*

For in vivo testing, mice were divided into 7 groups, one control and 6 experimental groups that received "CN" which received the NCs. At this point, different administration routes of the NCs were investigated.

Under light microscopy, the liver in the control group is normal; the hepatocytes are large and cuboidal with a prominent round nucleus and eosinophilic cytoplasm. The cords are radially arranged from the centrilobular venule to the periphery of the lobule. Hepatic sinusoidal capillaries are arranged between the hepatic plates with a sparse arrangement of Kupffer cells. In the control group, the kidney has a normal appearance of both the cortex and medulla. Malpighian corpuscles and urinary tubules show no changes (Figure 10). Hematoxylin- and eosin (HE)-stained sections from the control group (group I) showed

,

normal histological architecture of the lungs: thin-walled alveoli, alveolar sacs, clear alveolar spaces, and thin-walled blood vessels. The epithelial lining of the alveoli was composed of squamous alveolar cells with dense nuclei (type I pneumocytes) and large alveolar cells with large, rounded nuclei (type II pneumocytes). The spleen in the control group is covered by a thin capsule of connective tissue and shows white pulp and red pulp without changes. In the liver, kidney, and lung, congestion of blood vessels was observed.

**Figure 10.** Histological structure of liver, kidney, lung, and spleen in control mice. HE staining.

In the case of the experimental groups, by HE staining, no major changes in the histological structure of the organs studied were observed (Table 5).

− − − − CN-4 and CN-6 produced reduced histological changes in the organelles studied depending on the route of administration and dose. Administration p.o. and s.c., irrespective of dose, caused ectasia of a small number of veins in the liver (Figure 11), kidney (Figure 12), lung (Figure 13), and spleen (Figure 14). In the liver the hepatocytes show microvesicles and an increase in the number of Kupffer cells (6–7.5/12,879.8 mm<sup>2</sup> ). In the lung there is a high number of alveolar macrophages residing in the septal wall (14–24/12,879.8 mm<sup>2</sup> ). The alveoli have an intact, thin septal wall, but in some places it is thickened by macrophages. In the kidneys, the proximal convoluted tubules show rare brush border changes, and a small number of Malpighian corpuscles have a condensed glomerulus and increased capsular space volume (one in each preparation examined). In the interstitial space of the urinary tubules in the cortical area macrophages are observed (2–5/12,879.8 mm<sup>2</sup> ). In the spleen the lymphoid follicles show a slight increase, where pigments and megakaryocytes, some of them giant, appear.

− − − − − Administration of i.p. CN-4 0.01 mL and 0.02 mL in the liver, in hepatocytes with microand macrovesicles, resulted in 7–9.3 Kupffer cells /12,878.2 mm<sup>2</sup> , alveolar macrophages (9–13/12,879.8 mm<sup>2</sup> ), and interstitial macrophages between the urinary tubules 4–6.6/12,879.8 mm<sup>2</sup> . A small number (1–3 out of 10 fields examined of each individual) of Malpighian corpuscles were found with sclerotic changes of the glomerulus, with dilated convoluted tubules and with damaged brush border. Administration of i.p. CN-6

− − −

− − −

0.01 mL and 0.02 mL caused ectasia of some veins in the liver, kidney, and lung. The 0.02 mL dose produced changes in hepatocytes represented by macrovesicles, small focal necrosis, frequent Kupffer cells (9–11/12,879.8 mm<sup>2</sup> ), rare Malpighian corpuscles with sclerosing glomerular lesions (3 in each preparation examined), a reduced frequency of dilated proximal convoluted tubules with secretion in the lumen, macrophages in the interstitial space between the urinary tubules (5–7.5/12,879.8 mm<sup>2</sup> ), and alveolar macrophages in the septal wall of the pulmonary alveoli (9–18.5/12,879.8 mm<sup>2</sup> ). In the lung, clusters of 15–20 macrophages were observed in the lamina propria of the bronchioles and perivascular. − − −


− − − −

**Table 5.** Assessment of cellular and vascular changes produced in the organs under study.

Scoring of HE histological lesions was done by assessing changes and scoring as follows: no change (−), minor (+), medium (++).

**Figure 11.** Histological structure of the liver in experimental groups. HE staining.

*Polymers* **2022**, *14*, 1811

**Figure 12.** Kidney in experimental groups. HE stain.

From this figure it can be seen that in all experimental groups, the main change is in congestive blood vessels and a higher frequency of Kupffer cells. The arrangement of hepatocytes in radial cords is preserved. Cell shape and nuclei are similar to the cells in the control group. Hepatocytes from experimental groups receiving CN-4 and CN-6 p.o. have hepatocytes with intracytoplasmic microvesicles. In the groups administered s.c. and i.p., cytoplasmic micro and macrovesicles and focal necrosis are observed.

From this figure it can be seen that in all experimental groups the main change is in the vascular congestion. In the experimental groups that received CN-4 and CN-6 p.o., a reduced number of interstitial macrophages appear among the urinary tubules in the cortical area. In the groups given s.c. and i.p., the Malpighian corpuscles with glomerular compaction, modified brush border in some proximal convoluted tubules, and small hemorrhages in the cortical area are observed. Some tubules with increased nephrocytes in volume were apparent.

**Figure 14.** Spleen in the experimental group. HE staining.

Sections from the experimental groups showed preserved lung architecture. CN administration produced at some sites a change in alveolar septal diameter due to the accumulation of alveolar macrophages. Alveoli in the vicinity of congested vessels were observed in collapse. The number of alveolar macrophages located in the angular septal wall between alveoli was higher upon p.o. and i.p. administration. In the i.p. administration groups, macrophage clusters were observed in the lamina propria of the bronchi or perivascular. No alveolar macrophages were observed in the alveolar lumen.

In the experimental groups, the number of lymphoid follicles is increased, especially when administered i.p.; lymphatic cords have a slightly increased volume, and the number and frequency of megakaryocytes and pigment cells is higher in the experimental groups compared to the control group.

Following the evaluation of the IHC images, a low positive CD-4 label was observed for all markers regardless of the route of administration. Positive labeling was recorded for CN-6 only at intraperitoneal administration.

The CD147 marker was positive in the liver on Kupffer, endothelial, and stellate cells; in the kidney on endothelial and interstitial macrophages and mesangial and nephrocyte base cells; in the lung on alveolar macrophages and endothelial cells; in the spleen on endothelial lymphocytes and activated macrophages, platelets, and megakaryocytes. In the groups with intraperitoneal administration of CN-6, this CD147 marker was also expressed on hepatocytes around the centrilobular venule, on the nephrocyte basement, on a higher number of lymphocytes in the spleen, and on endothelial cells and type I pneumocytes in the lung. Although the IHC profile is positive in these organs, no pathological changes were observed (Figures 15–18).

In experimental groups receiving the CN-4 sample by various administration routes, P65 is expressed on endothelial cells in the liver, very little on endothelial cells in the lung, on nephrocyte bases and endothelial cells in the kidney, and on some lymphocytes and lymph nodes. Positive labeling was recorded in organelles from CN-6 with intraperitoneal administration (Figures 15–18).

α-SMA positively labelled blood vessel smooth muscle fibers in all organelles examined, some lymphocytes, endothelial cells, and red blood cells in capillaries. No excess

**Figure 15.** IHC marking in the liver.

**Figure 16.** IHC labeling in kidneys.

**Figure 17.** IHC labelling in the lung.

*Polymers* **2022**, *14*, 1811

MHC-II antigens were expressed on some lymphocytes (LB); monocytic lineage cells; a small population of T helper cells; activated T cells; arteriolar, sinusoidal, and venous endothelium; Kupffer cells; spindle cells in the connective tissue of the portal tract; large

hepatic veins; and liver capsule with the exception of the liver where duct cells were not labelled with MHCII (Figures 15–18). MHC II is expressed on bronchial epithelium, type II pneumocytes, and ciliated epithelial cells.

Cox-2 labeling was noted on cells of the monocytic macrophage system, namely Kupffer cells, splenic macrophages, alveolar macrophages, mesangial cells in kidneys, and at the base of nephrocytes (Figures 15–18).

All antibodies taken in the study label endothelial, stellate, and Kupffer cells in the CN-4 exposed batches. The batches exposed to CN-6, intraperitoneal administration also have hepatocytes positively labelled to CD147, p65, αSMA, MHC II, and COX-2. Positive labelling was also recorded in the erythrocytes in capillaries and venules.

The antibodies taken in the study mark endothelial cells, the membrane of the basal pole of nephrocytes, which has multiple invaginations which confers more intense positivity. Cox-2 and MHCII can also distinguish positively labelled mesangial cells. Higher positivity is recorded in the experimental batch CN6 intraperitoneal with both doses.

In all experimental groups, the number of alveolar macrophages detected by Cox-2 increased. Positive and low-positive labelling was also influenced by the positivity of hematomas in septal capillaries and blood vessels.

Low positive labeling CD147, p65, MHC II, and Cox2 was recorded on activated lymphocytes and macrophages in the CN-4 group. Positive MHCII and Cox-2 labelling was recorded in all experimental groups.

NPC has been successfully used for mucosal administration such as oral, nasal, ocular, and pulmonary, due to its mucoadhesive and mucosal permeability properties [19,20]. NPCs have been shown to bind efficiently to intestinal epithelial cells and effectively cross the epithelial barrier to penetrate tissues. This suggests that it is possible that NPCs are taken up by antigen-presenting cells of the mucosal immune system which subsequently transport the delivered antigens to immune initiation sites, such as Peyer's patches, to induce an immune response [21].

The presence of mononuclear phagocyte system macrophages in the liver, lung, kidney and spleen upon per o.s. administration in our experiment underlines this property of stimulation and distribution by macrophages in the body of chitosan nanoparticles. Following per o.s. and s.c. administration, a significant reactivity was achieved in the lung, followed by the liver, spleen, and kidney. In addition, the i.p. administration induced increased numbers of splenic lymphoid follicles and a slight increase in the number of macrophages in the interstitial space of the urinary tubules in the cortical area of the kidney.

Kupffer cells are specialized in the internalization of foreign nanoparticles, playing an essential role in their uptake, trafficking, and destination in the body (He et al., 2010). After intravenous administration, chitosan nanoparticles are opsonized in the blood before being phagocytosed by macrophages and accumulating in cells of the mononuclear phagocyte system. This passive targeting promotes the accumulation of NPs in the liver [22]. Transport from Kupffer cells to hepatocytes was also studied in mice, and it was observed that the majority of CsNps were localized in hepatocytes after intravenous injection. Micro and macrovesicles in hepatocytes could be given by the accumulation of chitosan nanoparticles in these cells in our experiment.

Kupffer cells internalize NP via several toll-like receptors, mannose, and Fc (Gustafson et al., 2015). The mechanisms involved are macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and the additional endocytotic pathway [23–25]. Clathrinmediated endocytosis has been shown to be responsible for the internalization of approximately 100–350 nm size, while caveolin-mediated endocytosis is responsible for endocytosis of 20–100 nm size [26–28]. Macropinocytosis enables internalization of 0.5–5 µm nanosystems [29].

The liver is a major site of accumulation of CS nanoparticles after intravenous administration [30–32]. Administration of CNP by various routes did not produce major histological changes in the organs studied, but there was a proliferation of liver Kupffer cells and alveolar macrophages in the septal space of the pulmonary alveoli without changing

its diameter. Intravenous administration of NCs does not cause significant hemodynamic changes, and 30 min after administration of NCs, they accumulate mainly in the liver and lung without causing hemolysis and leukocytosis [33]. The toxicity of CS nanoparticles was manifested by a short-term delay in weight gain as reported in the literature in rats. We did not encounter granulomas in the liver as reported by other researchers [33]. Granulomas found in the lung and liver indicate slow biodegradation of chitosan nanoparticles. Overall, the results obtained indicate good tolerability of intravenous administration of an unmodified chitosan suspension in the dose range studied [33]. The effect of chitosan on the viability of hepatocytes has been investigated in a series of studies on the creation of artificial livers in which chitosan served as a framework (nanofibre scaffold) for hepatocyte cultures. The presence of CS fibres in the intercellular space, performing a supportive function, improved the function of hepatocytes [34,35].

The biocompatibility of NCs is also explained by the fact that, under in vivo conditions, negatively charged plasma proteins are adsorbed on the surfaces of nanoparticles, thus preventing erythrocyte aggregation and hemolysis. The process of thrombosis during systemic NC incorporation is directly dependent on the magnitude of positive NCs' charge. Thus, pronounced agglutination, hemolysis, and intravascular thrombosis develop in the case of NCs with high charge and high concentration [36], whereas NCs with low charge adsorb coagulation factors, mainly fibrinogen, upon contact with blood and cause only weak inhibition of platelet aggregation [37]. In addition, the charge of NCs and their derivatives depends on the pH of the medium and is determined by the concentration of amino groups in the polymer molecule [38,39]. Resuspension in saline reduces NCs load, thus allowing intravenous administration of those without the risk of hemolysis [38] at particles doses of 4–6 mg/kg [40–42]. Including s.c. and i.p. administration was possible in the present experiment without thrombosis or hemolysis phenomena. Lee et al. [43] suggest that the lungs may be the primary barrier organ for intravenously administered CPN. This phenomenon has already been described and has, in fact, been proposed as a targeted delivery strategy for the lungs [43]. Proliferation of alveolar macrophages was also observed in our experiment regardless of the route of administration.

After entering the body, NMs (>6 nm) predominantly accumulate in organs of the mononuclear macrophage system (MPS), such as the liver, spleen, lungs, etc. [40], due to subsequent recognition and internalization by the macrophages of this system [44]. Some particles, such as gold NPs (40 nm) and [45] dextran-coated magnetite NPs (core diameter of 8–10 nm) [46,47] are gradually degraded in cells over several months or even longer periods and sequestered in MPS for a long period of time. Indeed, it has been observed that long-term sequestration in MPS induces some potential side effects, especially immunotoxicity [48]. Accordingly, removal of NCs from MPS organs, especially the liver, sequesters 30–99% of administered NCs from the bloodstream [40] being essential for the clinical safety of NCs. Therefore, it is necessary that all injected NCs are completely removed within a reasonable period of time [49]. Following distribution in hepatocytes, the hepatobiliary–fecal excretion route has been shown to be the primary elimination pathway for CsNps in addition to the reno-urinary excretion pathway. Elimination of CsNps in mice was a lengthy process with a half-life of approximately 2 months.

CD147, a transmembrane glycoprotein with two immunoglobulin-like glycoproteins of the immunoglobulin G (IgG) superfamily, is widely expressed on the surface of various cells, activated lymphocytes, and epithelial cells including cancer cells. As a metalloproteinase inducer, CD147 has been shown to be involved in multiple biological processes such as immune response, tumour progression, and tissue repair. CD147 is recognized as a regulator of lipid metabolism in a variety of cell types and autophagy [50]. In experimental groups, this marker was captured on various cells, but no histopathological changes were reported. Modified CS nanoparticles activate macrophages and are phagocytosed by them [51], processes that lead to aseptic inflammation [38,52]. CD147 is expressed by platelets and is overexpressed following platelet activation [53]. The CS nanoparticles used in this experiment have a small cytotoxic effect and have a weak antiplatelet and

anticoagulant effect as mentioned by Sonin et al. [33]. CD147-mediated cell–cell interaction on the cell surface induces platelet activation and via NF-B-factor-mediated monocyte activation will occur [54].

Nuclear factor-κB (NF-κB/p65) is a family of transcription factors that plays a critical role in inflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-κB activation depends on proteosomal degradation induced by phosphorylation of NF-κB inhibitory proteins (IκB), which retain inactive NF-κB dimers in the cytosol in unstimulated cells. Most of the signalling pathways leading to NF-κB activation converge on the IκB kinase complex (IKK) which is responsible for IκB phosphorylation and is essential for signal transduction to NF-κB. Further regulation of NF-κB activity is achieved by various posttranslational modifications of the basic components of NF-κB signalling pathways. In addition to cytosolic modifications of IKK and IκB proteins, as well as other pathway-specific mediators, transcription factors are themselves extensively modified [55]. The low positive labeling after CN-4 administration demonstrates the presence of modified transcription factors not involved in transcriptional processes. In animals exposed to CN-6, positive labeling may indicate involvement of this factor in transcription. Numerous labelled cells were observed in the spleen of mice from experimental groups. Macrophages are able to integrate an impressive amount of information on the identity and virulence of pathogens, as well as endogenous cues present in their microenvironment, to modulate the immune response for optimal host protection. Central to this capacity are the numerous ways in which NF-κB signaling is modulated based on shifting activation thresholds, integration of information from different classes of pattern recognition receptors, and tight regulation of transcription through rigorous positive and negative feedback loops [54]. The presence of this p65 marker was low positive in the studied organelles with the exception of the CN-6 intraperitoneal exposed batches, denoting a reduced inflammatory response.

Circulating monocytes are recruited into tissues, where they differentiate into macrophages and take part in the process of inflammation or tissue remodeling. According to the traditional concept, macrophages are classified into pro-inflammatory (M1), non-activated (M0), or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. More recent experimental findings have led to a substantial update of the monocyte–macrophage nomenclature to include the nature of the polarization signal. In response to proinflammatory stimuli, monocytes can be polarized directly into three subsets of macrophages with M1-type proinflammatory phenotype; interferon-γ-induced macrophages have the strongest proinflammatory properties. When exposed to various anti-inflammatory stimuli, monocytes can differentiate into at least five subsets of M2-type macrophages. Of these, a subset generated upon exposure to IL-4 (IL-13) has the most typical M2-type characteristics. In both humans and mice, differentiation of monocytes into macrophages involves global transcriptome changes that are tightly controlled by various transcriptional regulators and signaling mechanisms [56]. There are many key checkpoints in the transcriptional control and signaling network that trigger either pro-inflammatory or anti-inflammatory polarization [57]. Regulation of NF-κB family transcriptional activity plays a central role in M1–M2 switching and macrophage polarization towards an anti-inflammatory or pro-inflammatory phenotype. The absence of intense p65 labeling also implies insignificant macrophage activation in this experiment.

MHC-II or HLA-DR is a molecule involved in antigen presentation and is the main signal in immunological cooperation [58]. MHC-II antigens are normally expressed on B lymphocytes, monocytic lineage cells, a small population of T helper cells, activated T cells, thymic epithelium, and vascular endothelium [59]. Normal arteriolar, sinusoidal, and central venous endothelium often express MHC II. Kupffer cells have always expressed these antigens. MHC II-positive spindle cell fibroblasts were identified in the connective tissue of the portal tract, large hepatic veins, and liver capsule: most shared antigens common to all leukocytes and reacted with MHC II. Bile duct epithelium expressed MHC II in primary biliary cirrhosis, large duct obstruction, and drug-induced cholestasis, indicating that MHC II positive spindle cells are phenotypically similar to histiocytes. In our experiment liver

duct cells were not labeled with MHCII. Although Kupffer cells express lower levels of MHC class II molecules than classical dendritic cells, they are able to interact with T cells. However, unlike dendritic cells, Kupffer cells favor the development of regulatory T cells, thereby promoting immune tolerance [60] which explains the lack of liver injury in this experiment following NC administration. Hepatic macrophages may be highly targeted by nanoparticle drug carriers due to their efficient phagocytosis function in the liver [61].

MHCII is expressed on both bronchial and alveolar epithelium, especially on type II pneumocytes and ciliated cells, and in this experiment as reported in the literature [58].

Various studies have shown that CS and its derivatives can effectively activate antigenpresenting cells and induce cytokine stimulation to produce an effective immune response and promote Th1/Th2 response balance [62]. Catalytically active Cox-2 (and Cox-1) is located in the nuclear envelope (NE) and endoplasmic reticulum (ER), where it mediates PGE2 biosynthesis. Cox-2 dissociated from the nuclear envelope is catalytically inactive [63]. Activated macrophages overexpress these enzymes (Cox-2), which would lead to the production of large amounts of PGs. In addition, NF-κB is a transcription factor that induces copying of proinflammatory genes to produce large amounts of proinflammatory mediators, such as Cox-2, in activated macrophages [55]. The presence of a positive low label at this marker indicates reduced transcription of proinflammatory mediators. Although a large number of alveolar macrophages can be observed in the lung, low positive for alpha SMA indicates a lack of synthesis of collagen precursors. This is also observed in other organs.

Cyclooxygenases are responsible for the synthesis of prostaglandins from arachidonic acid and are present in two isoforms in the kidney, Cox-1 and Cox-2. Cox-1 is involved in the regulation of basic cellular functions, while Cox-2 is a proinflammatory enzyme and is induced by inflammatory stimuli [64]. Cox-2 labeling has been noted in cells of the monocytic macrophage system, namely Kupffer cells, splenic macrophages, alveolar macrophages, and mesangial cells in the kidney. The absence of an inflammatory process denotes that Cox-2 is in an inactive phase.

The adjuvant effect of CS is mainly evaluated from several points of view such as biocompatibility, biodegradability, and cell permeability [65]. Size and surface charge are key characteristics that define how cells interact with and internalize NCs [66]. The affinity of NCs to the cell membrane is related to the cationic component, a characteristic of CS [67].

Upon oral administration an important role in nanoparticle uptake is played by M cells in the intestinal mucosa. The key point for the initiation of the mucosal immune response is antigen uptake, in the case of the NCs based on CS. Many experiments have shown that M cells can carry various macromolecular substances and microorganisms. After M cells in Peyer plates adsorb NCs, they are actively transported to the underlying immune cells, dendritic cells, to stimulate the local immune system or mucosal immunity [68]. Previous research has shown that CS nanoparticles accumulate predominantly in the macrophages of the monocytic macrophage system [69]. Administered systemically, nanoparticles are taken up by Peyer's plaque cells, pass into the lymph and then into the general blood circulation, and can subsequently be taken up by the liver, kidney, spleen, heart, and other vital organs [70]. CS nanoparticles are also known as immunomodulators. An important role in this case is played by the size and physicochemical characteristics of the particles that can influence the interaction with immune cells to induce the desired therapeutic benefit [65]. Bioactive CS nanoparticles are internalized by macropinocytosis, clathrinmediated endocytosis, and phagocytosis. They are then transported intracellularly by endosomes, multivesicular bodies, and lysosomes. Proteomics elucidated that chitosan nanoparticles induced an increase in proteins involved in immunoregulatory functions and antioxidant activities. They also promoted the production of anti-inflammatory/proregenerative mediators but suppressed pro-inflammatory ones. Therefore, CS nanoparticles could prevent persistent post-treatment inflammation [71].

Most research that has addressed the toxicity of chitosan-based nanoparticles has typically conducted biocompatibility studies 2–4 days after intravenous administration

in animal models [72–75]. However, the toxicity determined from these types of studies, which use short observation periods for long-circulating dispersed solutions [72,74], may not be fully representative because neither biodistribution nor biodegradation processes are completed within these short periods. However, studies over longer observation periods after intravenous administration in subacute or chronic experiments are rare [33], even though long-term observations of biological effects of dispersed systems are no less important than acute toxicity analysis. Based on this consideration, we chose chronic exposure with per o.s., s.c., and i.p. administration.

#### **4. Conclusions**

Interfacial condensation of CS with poly(NVPAI) is an effective method of obtaining NCs capable of encapsulating, transporting, and delivering drugs. The morphological characteristics of NCs as well as their physicochemical properties can be modeled by varying some parameters of the preparation process (the ratio between the functional groups of the polymers involved in the reaction, the ratio of the phases in which the polymers are solubilized). Consistent with these properties also varies the ability of NCs to encapsulate and release the model drug in a simulated physiological environment (pH = 7.4).

Low positive labeling at all markers taken in the study in experimental groups subjected to CN-4 sample denotes biocompatibility and does not result in histological changes. These NCs have demonstrated the ability to stimulate cells of the mononuclear phagocyte system in the liver, lung, spleen, and kidney without producing inflammatory changes. When administered per o.s., proliferation of alveolar macrophages was observed, followed by Kupffer cells. Administration of CN4 and CN6 i.p. at a dose of 0.02 mL induced microand macrovacuoles of hepatocytes. Overexpression of CD147, p65, MHCII, and Cox-2 markers was observed in the macrophages of the organs studied in all experimental groups. The absence of alpha SMA labelling denotes the absence of an inflammatory process.

From this study, it can be concluded that the obtained NCs have the advantage of being biocompatible, non-toxic, and safe for in vivo administration as drug delivery systems.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14091811/s1, Figure S1: Evolution of mice bodyweight through the experiment (the first measurement was made in the first day of the experiment, the second one at 10 day and the last one at the final of the experiment); Table S1: *p*-values of Student's *t*-test for simple 5-FU compared with 5-FU loaded NCs.

**Author Contributions:** Conceptualization, K.Z.D., D.M.R., A.N.C., M.P. and L.I.A.; methodology, K.Z.D., M.D., D.M.R., A.N.C., L.I.A., M.-C.S. and C.S.; software, D.M.R., A.N.C., M.-C.S. and C.S.; investigation, K.Z.D., D.M.R., A.N.C., M.-C.S. and C.S.; writing—original draft preparation, D.M.R., M.P., L.I.A. and C.S.; visualization, D.M.R., A.N.C., L.I.A., M.P. and C.S.; supervision, M.P., L.I.A. and C.S.; project administration, L.I.A. and M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research leading to these results has received funding from the NO Grants 2014–2021, under project contract no. 15⁄2020.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


## *Review* **Paclitaxel Drug Delivery Systems: Focus on Nanocrystals' Surface Modifications**

**Razan Haddad 1, \* , Nasr Alrabadi 2, \* , Bashar Altaani <sup>1</sup> and Tonglei Li 3**


**Abstract:** Paclitaxel (PTX) is a chemotherapeutic agent that belongs to the taxane family and which was approved to treat various kinds of cancers including breast cancer, ovarian cancer, advanced non-small-cell lung cancer, and acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma. Several delivery systems for PTX have been developed to enhance its solubility and pharmacological properties involving liposomes, nanoparticles, microparticles, micelles, cosolvent methods, and the complexation with cyclodextrins and other materials that are summarized in this article. Specifically, this review discusses deeply the developed paclitaxel nanocrystal formulations. As PTX is a hydrophobic drug with inferior water solubility properties, which are improved a lot by nanocrystal formulation. Based on that, many studies employed nano-crystallization techniques not only to improve the oral delivery of PTX, but IV, intraperitoneal (IP), and local and intertumoral delivery systems were also developed. Additionally, superior and interesting properties of PTX NCs were achieved by performing additional modifications to the NCs, such as stabilization with surfactants and coating with polymers. This review summarizes these delivery systems by shedding light on their route of administration, the methods used in the preparation and modifications, the in vitro or in vivo models used, and the advantages obtained based on the developed formulations.

**Keywords:** paclitaxel; nanocrystals; surface modification; chemotherapy; cancer; drug delivery; nanotechnology

#### **1. Introduction**

Currently, cancer is considered a serious disease that is globally widespread, and it is one of the most life-threatening illnesses [1], accounting for about 10 million deaths in 2020 [2]. Additionally, the economic burden of this disease is enormous, and it is anticipated to increase in the future [3,4]. On the other hand, chemotherapeutic agents are considered effective at fighting cancer and preventing its development and progress [5]. However, there is still an urgent need for more therapeutic options or strategies to improve the currently available treatments in terms of safety and efficacy.

The improvements of chemotherapeutic agents mainly depend on two research lines [5]. The first one is related to explaining cancer-specific mechanisms and molecular targets, such as signal transduction inhibitors concerning essential processes of cells such as growth, survival, and differentiation. These substances may have the ability to prevent the injuries caused by cancer cells, including proliferation and tissue invasion [6]. The second line is considering the enhancement of the available cytotoxic drugs which act on abundant targets (e.g., DNA or tubulin) [5]. These cytotoxic drugs are either natural products or their derivatives obtained from plants, marine species, and microorganisms,

**Citation:** Haddad, R.; Alrabadi, N.; Altaani, B.; Li, T. Paclitaxel Drug Delivery Systems: Focus on Nanocrystals' Surface Modifications. *Polymers* **2022**, *14*, 658. https:// doi.org/10.3390/polym14040658

Academic Editor: Leonard Atanase

Received: 19 December 2021 Accepted: 3 February 2022 Published: 9 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

but unfortunately, these drugs are still toxic to normal cells [7]. Therefore, the improvement of their efficacy and safety is always warranted.

Eventually, many anticancer agents were obtained. but most of them are inefficient and cause severe side effects. Therefore, there is an emerging need to develop new therapeutic agents or delivery approaches. Several drug delivery systems based on nanotechnology modalities have been obtained for different anticancer drugs such as solid lipid nanoparticles, liposomes, micelles, polymeric nanoparticles, nano-emulsions, implants, and nanocrystals [8–10]. All these approaches are aimed at either enhancing the efficacy or reducing the side effects of the currently available chemotherapeutic agents. Finding novel and appropriate drug delivery systems is crucial, especially for chemotherapies where intravenous delivery remains the main route used for drug administration [8]. This returns to the fact that most anticancer drugs have low solubility or gastrointestinal tract (GIT) toxic side effects over oral administration, which in turn can reduce their oral absorption below the therapeutic effective levels [10].

#### **2. Paclitaxel**

Paclitaxel (PTX) is an important chemotherapeutic agent that belongs to the taxane family. Taxanes were initially obtained from plants of the genus *Taxus*. PTX was first derived from the bark of the Pacific yew (*Taxus brevifolia*), which is an evergreen tree and small to medium in size and also known as western yew, native to the Pacific Northwest of North America [11,12]. PTX was approved by the United States (US) Food and Drug Administration (FDA) to treat various kinds of cancers including breast cancer, ovarian cancer, advanced non-small-cell lung cancer, and acquired immunodeficiency syndrome (AIDS) related Kaposi's sarcoma [13]. In general, PTX is not well tolerated and related to serious adverse drug effects such as hypersensitivity reactions, hematological toxicity, peripheral sensory neuropathy, and myalgia or arthralgia [13], even though PTX has been used for two decades either as a single drug or in combination with other chemotherapeutics.

The antitumor activity of paclitaxel comes from its high binding affinity to microtubules, stabilizing and improving the polymerization of tubulin and destruction of the dynamics of the spindle microtubule [14,15]. Such activities provide effective inhibition of cell mitosis, intracellular transport, and motility, which end up with cell death by apoptosis. However, the clinical developments of the natural form of paclitaxel have been restricted due to its physicochemical properties, particularly its very low solubility [16]. Additionally, the absence of modifiable functional moieties in its structure makes the chemical alteration of the natural paclitaxel very complicated when attempting to enhance its solubility [17]. Considering that, the selection of a proper delivery system to paclitaxel is considered very crucial to improving its clinical development, safety, and efficiency.

Regarding the chemical structure of PTX (Figure 1), its 20-carbon compound (C20) belongs to the diterpene class of natural compounds [18]. The anticancer activity is mainly recognized for ring A, ring D (the oxetane ring), the C2 benzoyl group, and some components such as the C3′ amide-acyl group and the OH group at C2′ , which attaches on the side chain to C13 [19]. On the other hand, other groups slightly affect the therapeutic activity of PTX such as the carbonyl group on C9 and the acetyl group on C10. Moreover, the specified conformation of the paclitaxel molecule is provided by the acetyl group [19].

PTX has poor aqueous solubility, low permeability, and as it is a P-gp substrate, it also has limited capabilities for being delivered via the oral route [20–23]. The poor permeability of PTX is related to the following molecular factors: its molecular weight is more than 500, the hydrogen bond acceptor (HBA) is greater than 10, and the polar surface area (PSA) is more than 140 A<sup>2</sup> , which results in a permeability coefficient value in the range of 10−<sup>6</sup> cm/s [24,25]. Therefore, PTX is administered parentally via the intravenous (IV) route with a suitable cosolvent (cremophor EL and ethanol), which unfortunately ends up with several direct, problematic, adverse effects such as acro-anesthesia and neurovirulence, causing pain and high cost [26–29].

**Figure 1.** The chemical structure of a PTX drug.

#### **3. PTX Formulations**

mixture of ethanol and Cremophor EL™ (a polyoxyethylated castor oil). Taxol – To improve the benefit and delivery of PTX, several formulations have been developed. The most commonly used delivery system is a cosolvent strategy based on a 50:50 mixture of ethanol and Cremophor EL™ (a polyoxyethylated castor oil). Taxol ® is the first generic product of paclitaxel, and it consists of this cosolvent mixture. Although this method overcomes the problem of solubility, Cremophor EL has been associated with non-linear pharmacokinetics and serious and dose-limiting toxicities, such as hypersensitivity, neurotoxicity, and nephrotoxicity [11]. Due to these adverse effects, Taxol ® is given slowly in 135 or 175-mg/m<sup>2</sup> doses by infusion over 3–24 h every 3 weeks [30,31].

Abraxane™ is another marketed drug of PTX, which was produced by Abraxis Bio- . Moreover, Abraxane™ has a linear pharmacokinetic profile and a higher in-Abraxane™ is another marketed drug of PTX, which was produced by Abraxis Bio-Science (later obtained by the Celgene company) and approved by the FDA in 2005 [32]. The formulation of PTX in this product is performed with human serum albumin (HSA) [33]. HSA is the most abundant plasma protein in the blood, with a large half-life that reaches up to 19 h and which can bind hydrophobic substances irreversibly, transport them through the body, and deliver them to the cell surface [34]. Additionally, HSA plays a significant role in cellular uptake and transcytosis, as it is bound to gp60 and other proteins which are highly expressed in malignant cells, such as secreted proteins acidic and rich in cysteine (SPARC). Nevertheless, it is still ambiguous how exactly HAS improved the biological response of PTX. However, it is significantly clear that the removal of Cremphor EL contributes to the ability to administer a higher dose of PTX with an analogous toxicity [35]. Moreover, Abraxane™ has a linear pharmacokinetic profile and a higher intratumoral concentration by 33% in comparison with Taxol ®, based on results obtained by the Abraxis BioSciences company [36].

Another marketed drug of PTX is Lipusu™, which was formulated by Luye Pharma- , Lipusu™ has similar activities toward Another marketed drug of PTX is Lipusu™, which was formulated by Luye Pharmaceutical Co. Ltd. and approved in China in 2003. It is a liposome composed of PTX, lecithin, and cholesterol. In comparison with Taxol ®, Lipusu™ has similar activities toward breast cancer, non-small cell lung, and gastric cancer but with considerably lesser side effects [37,38].

Finally, Genexol-PM™ is marketed by Samyang Corporation and was approved in South Korea in 2007. It is composed of PTX and poly (ethylene glycol)-b-poly (lactic acid) (PEG-b-PLA) block copolymers. Clinical studies showed that Genexol-PM™ has dosedependent pharmacokinetics and good tolerance, especially for patients with advanced pancreatic cancer or metastatic breast cancer [39–41]. PM™ has dose –

PM™ is marketed by Samyang Corporation and was approved in

#### **4. Drug Delivery of PTX**

As previously mentioned, the physicochemical properties and the nature of PTX complicated its formulations. Consequently, several delivery systems for PTX have been developed to enhance its solubility and pharmacological properties involving micelles, liposomes, nanoparticles, the prodrug approach, emulsions, implants, and nanocrystals [42–45]. Figure 2 summarizes the most common strategies utilized for PTX delivery systems. –

**Figure 2.** The most common developed strategies to improve the delivery of paclitaxel drugs. SLN: solid lipid nanoparticles.

#### *4.1. Micelles*

Generally, micelles consist of polar heads that encounter the outside aqueous environment and non-polar tails, which form the interior hydrophobic core. Above the critical micelle concentration, micelles spontaneously form, and drugs with low solubility encapsulate efficiently in the lipidic core [46,47]. The properties of micelles and the hydrophobic regions can be modified and tailored by using various polymer structures [48]. The targeted delivery of PTX micelles developed using an Asn-Gly-Arg (NGR) peptide, which covalently bonds to PEG chains to deliver PTX through a brain tumor [49]. Moreover, the oral delivery of PTX was obtained using multi-functional chitosan polymeric micelles [50]. The development of redox-sensitive PEG2000-S-S-PTX micelles resulted in a reduction of PTX cytotoxicity in ovarian and breast cancer cells [51].

#### *4.2. Liposomes*

The liposome is a spherical structure with a membrane composed of a single or multiple phospholipid bilayers. It has an aqueous core to encapsulate hydrophilic drugs, while the hydrophobic drug can be loaded in the region of the bilayer membrane. Liposomes have been used to deliver PTX because they showed that they can enhance solubility and efficacy by modulating its pharmacokinetic properties. Additionally, the used excipients are clinically approved [52]. LipusuTM is the first injected PTX liposome, and it has been used in China to treat non-small cell lung cancer, breast cancer, and other cancers [19]. It maintains the original activity of PTX but with a significant reduction in the side effects. Moreover, LEP-ETU is another liposome loaded with PTX. Based on the phase I study, LEP-ETU showed little difference in the pharmacokinetic properties in comparison to Taxol® while being safer at higher doses [53].

Long-term instability is the main obstacle related to liposomes. Despite liposomes having the ability to deliver cytotoxic compounds to certain tissues, they can be eliminated by the mononuclear phagocytic system (MPS) in the spleen and liver [54]. Interestingly, the average circulation time of liposomes can be enhanced by 10 folders with PEGylation, which results in an improvement of the half-life of PTX and its antitumor properties [55–57]. The PEGylated liposome can be also modified by active targeting strategies to improve its efficacy [58,59]. This can be obtained by covalently binding species to the surface of the liposomes, such as the peptides [60], proteins [61], and tissue-specific antibodies [62]. Specifically, a multifunctional peptide was incorporated to the surface of the liposomes loaded with PTX, which improved its targeting activity and also its efficacy [59]. Moreover, triphenylphosphonium (TPP) was incorporated into the surface of the PEGylated PTX liposomes, which consequently enhanced their cytotoxicity and antitumor efficacy and provided efficient mitochondrial targeting in cancer cells [58]. Moreover, PTX loaded to a pH-sensitive lipid that was incorporated into a liposomal membrane prevented liposome degradation by lysozymes and consequently caused more suppression of tumors by providing more PTX accumulation at pH of 7.4 instead of 5.5 [63,64].

#### *4.3. Nanoparticles*

#### 4.3.1. Solid Lipid Nanoparticles

Generally, solid lipid nanoparticles (SLN) are obtained from solid lipids, such as complex glyceride, highly purified triglycerides, and waxes [65]. Several kinds of lipids and surfactants can be used for SLN production and engineering. More specifically, lipids such as phospholipids and glycerides and surfactants such as tween 80, sodium glycolate, lecithin, and poloxamer 188 are considered suitable for IV injection [66]. There are several advantages related to the SLN, such as the simplicity of the preparation method and the scaling up, biocompatibility, stability, low cost, low toxicity, controlled drug release, and versatile chemistry [52].

To obtain high drug loading and the slow release of PTX, SLN should obtain high drug solubility and miscibility [67]. The cellular uptake and cytotoxicity properties of PTX-loaded SLN can be vary based on the lipid materials used. For instance, studies showed that the cellular uptake of SLN was concentration- and time-dependent and related to the melting point of the lipidic materials, the length of its hydrocarbon chain, and the particle size [68–70]. PTX-loaded PEGylated steric acid SLN proved to have a high cellular uptake and up to 10-fold greater cytotoxicity in comparison with PTX. Moreover, SLN showed an ability to affect P-gp-mediated multidrug resistance (MDR), as PTX loaded SLN provided an inhibition of P-gp activity and a rapid depletion of ATP [71,72].

As with the other noncompaction systems, surface modification of the particles by different chemical moieties is useful for obtaining prolonged SLN circulation by avoiding the clearance with the reticuloendothelial system (RES) [52].

#### 4.3.2. Polymeric Nanoparticles

Polymers have been used in nanoparticle preparation to provide them with suitable properties and characteristics. Examples of some of the polymers that are commonly used in developing paclitaxel nanoparticles are poly (lactic-co-glycolic acid) (PLGA) and chitosan, which will be discussed in the following sections.

#### Poly Lactic-co-Glycolic Acid (PLGA)

Poly (lactic-co-glycolic acid) (PLGA) is a biocompatible, biodegradable, nontoxic synthetic polymer derived from poly (lactic acid) (PLA) and poly (glycolic acid) (PGA) [73,74]. It has been approved by the US Food and Drug Administration (FDA) for drug delivery, as it has superior properties in the delivery of many therapeutic agents. PLGA is a very useful and successful polymer in nanomedicine and the nano-delivery of drugs. In addition, it has a favorable ability to target tumors and DNA [73,75–77]. PLGA is available commercially with various molecular weights and copolymer ratios. Based on that, the duration of the degradation can vary, as can the release time. Glycolic acid is more hydrophilic than lactic acid, and thus PLGA with higher glycolic acid is more hydrophilic and can adsorb water more and degrade faster [78,79]. The loading of PTX to PLGA nanoparticles has been obtained by various methods such as emulsion solvent evaporation [77], interfacial deposition methods [80], and the nanoprecipitation method [81]. Studies showed that PTX loaded to the PLGA nanoparticles had superior antitumor properties and efficacy in comparison with Taxol® [81,82]. Moreover, surface modification of the nanoparticles has a crucial impact on their properties, such as efficacy and targeting. The delivery of PTX was improved by surface modification of PLGA nanoparticles with albumin, as the circulation time of these nanoparticles in the blood was increased even as it became more toxic in the in vitro study [83]. The targeted delivery of PTX to breast cancer cells was developed by loading it into PLGA nanoparticles coated with hyaluronic acid (HA), and the results showed that the cellular uptake was increased using this system [84]. Moreover, PTX has been loaded to lipid PLGA hybrid nanoparticles, and the results showed that the release profile was affected with this lipid coat. Also, these nanoparticles provided a prolongation in the circulation time in the blood [85].

#### Chitosan

Chitosan is a natural polysaccharide polymer produced by the diacylation of chitin. It has many attractive properties such as non-toxicity, biocompatibility, biodegradability, and bio-adhesivity, which necessitates its use in drug delivery [86,87]. The solubility of chitosan in acidic solutions and its limited solubility in biological solutions (pH 7.4) are considered the main drawbacks of its application in drug delivery. Lately, many chitosan derivatives have been prepared by adding various hydrophobic or hydrophilic groups to the chitosan structure [88,89]. Moreover, studies showed that chitosan has antitumor properties, and it can affect the cancer cells by interfering with its metabolism, inhibiting its growth, or inducing its apoptosis [90].

Chitosan has been introduced to many PTX delivery systems, and it improved various aspects (e.g., decreasing the toxicity and enhancing the efficiency and targeting capabilities) [91–93]. A study showed that the PTX-loaded micelle based on N-octyl-O-sulfate chitosan (OSC), which is a novel derivative of water-soluble chitosan used for the delivery of PTX, has superior toxic properties, as lower side effects were observed, and the AUC was about 3.5 lower than the marketed drug Taxol® while preserving the antitumor efficacy at equivalent doses [94]. Additionally, other studies showed that the targeted delivery of PTX

chitosan nanoparticles had been achieved in combination with other polymers such as PE-Gylated chitosan nanoparticles grafted with Arg-Gly-Asp (RGD) [92], poly NIPAAm [95], transferrin [96], and biotinylated N-palmitoyl chitosan [97].

#### *4.4. Prodrug Approach*

Prodrugs are derivatives of a drug molecule that can be transformed chemically or enzymatically in the body to release the active ingredient that possesses pharmacological effects [98]. Differing from other delivery systems or formulations, prodrugs are usually formulated by chemical linkage with proper quality control and less variation from batch to batch. Generally, prodrugs are developed to overcome problems related to the parent drug itself, such as poor aqueous solubility, limited permeability, inadequate oral absorption and delivery, non-targeting, and toxic side effects [99,100].

The PTX prodrug is usually fabricated at the carbon no. 123; 2′or 7-OH group [100]. PTX prodrugs are constructed using various strategies, such as polymer-based prodrugs, which are formulated using polymers such as PEG [101], PLA [102–104], poly(amidoamine) [PAMAM] [105], N-(2-hydroxypropyl) methacry'lamide (HPMA) [106], and poly(L-glutamic acid) (PGA) [107]. Moreover, a protein-based prodrug of PTX has been developed using different proteins, such as the marketed product Abraxane™, which is tumor-targeted and formulated using CREKA and LyP-1 [108]. Additionally, PTX prodrugs were obtained using transferrin (Tf) and Fmoc-L-glutamic acid 5-tert-butyl ester (linker) to specifically target tumor tissues and cells [109]. Similarly, peptide-based prodrugs of PTX were also formulated such as the Tat-based self-assembling peptide, which is used to deliver PTX intracellularly [110], the tumor-homing cell-penetrating peptide (CPP) [111], and recombinant chimeric polypeptides (CPs) [112]. Additionally, PTX prodrugs can be obtained using small molecules such as docosahexaenoic acid (DHA) [113], conjugated linoleic acids (CLAs) [114], and oligo(lactic acid)<sup>8</sup> [115]. Finally, hybrid prodrugs for PTX also exist, which are a combination of two drugs or more that is capable of producing synergistic effects, reducing the adverse effects related to a high dose of a single drug, and overcoming the multidrug resistance mechanism of cancer cells during treatment [116]. PTX hybrid prodrugs were delivered using other anticancer drugs such as doxorubicin (DOX) [117], camptothecin (CPT) [118], and the nucleic acid oligonucleotide [119].

#### *4.5. Emulsions*

Generally, macroemulsions are defined as the dispersion of one liquid in another liquid and it is considered a two-phase system [120]. They are turbid or opaque, viscus, and thermodynamically unstable, and their preparation is complicated as sheer is needed. On the other hand, microemulsions are translucent, thermodynamically stable, have a lower viscosity, and form spontaneously [121]. Based on the name, nano-emulsions should have a droplet size lower than microemulsions. As a matter of fact, nano-emulsions have a droplet size of 20–200 nm and a narrow particle size distribution [122–124].

A TocosolTM nano-emulsion was established early in 2000. It was formulated using an a-tocopherol isomer of vitamin E as a solubilizing agent for PTX and vitamin E TPGS as an emulsifier. Unfortunately, studies in phase III showed that the overall response rate was only 37%, while it was 45% with Taxol®. Based on that, the TocosolTM nanoemulsion was terminated [125]. Recently, Shakhwar et al. tried to reform a TocosolTM nanoemulsion using the c-tocotrienol (c-T3) isomer instead of a-tocopherol and the PEGylated c-T3 surfactant instead of vitamin E TPGS. Their results showed that the reformulated PTX was more active toward pancreatic tumor cell lines than the previous formulation [126].

Moreover, self-emulsifying drug delivery systems (SEDDSs) and self-microemulsifying drug delivery systems (SMEDDSs) are combinations of the non-aqueous components of emulsions and microemulsions, respectively [127], such as oils, surfactants, and if present, cosurfactant or cosolvents. These mixtures can be readily dispersed when diluted with an aqueous phase (gastric fluids) in the body and then spontaneously emulsified to form fine oilin-water (O/W) microemulsions. This process can be sped up by slight mechanical agitation,

and in vivo, this can be obtained by gastrointestinal motility [18,122,128]. A novel SMEDDS was developed for oral delivery of PTX, and it was administered to patients with advanced cancer and compared with orally administered Taxol®. The SMEDDS was co-administered with cyclosporin A to inhibit P-gp and CYP3A4. This formula was safe and well-tolerated by patients and had comparable bioavailability to oral Taxol ®. In addition, the T-max of the SMEDDS was lower than the orally delivered Taxol®. This means that the absorption was higher in the novel formula, and this may be related to the added excipients [129]. In another study, the oral delivery of PTX was designed as an SEDDS. In this study, tocopheryl polyethylene glycol succinate was used to assist the emulsification. The results indicated that this system had higher G2M cell cycle arrest, apoptosis, mitochondrial membrane potential disruption, and ROS production in comparison with Taxol®. Moreover, the oral bioavailability of the SEDDS was about fourfold greater than Taxol®. Considerable reductions in the volumes and weights of the tumors were detected in syngeneic mammary tumors in SD rats. Additionally, this system was safe, stable, and caused low lung metastasis [130].

#### *4.6. Implants*

Drug-loaded polymeric implants are considered a pioneering approach in drug delivery. Active ingredients can be delivered to malignant cells using biodegradable polymers in continuous, sustained, and predictable patterns. Owing to their nature, biodegradable polymers do not need to be removed surgically after their application and thus eliminate complications associated with the long-term safety of implanted devices with non-biodegradable polymers. Additionally, the postsurgical local insertion of a biodegradable implant device loaded with an anticancer drug can avoid the further spread of cancer cells while avoiding toxic chemotherapy adverse effects in the patient [131]. Recently, an in situ depot-forming implant (ISFI) has been developed which can be injected as a liquid and then subsequently solidified [132,133]. In this way, an effective dosage form can be delivered with the avoidance of surgical insertion [134]. Moreover, ISFIs have relatively simpler preparation conditions and fewer complications than solid implants [135,136]. The PTX ISFI was formulated using PLGA to improve its efficiency and toxicity. This formula provided an in vitro sustained release of PTX for 28 days [137].

#### *4.7. Nanocrystals*

Nanocrystal formulations have become more attractive for the delivery of chemotherapies due to their superior properties in comparison with other nano-delivery approaches [138–140]. Nanocrystals eliminate the need for chemical carriers, therefore eradicating any toxic side effects induced by the excipients used for solubilization or coating and also providing about 100% drug loading, which ensures suitable concentrations of the drug even at low doses [141]. Additionally, due to the stable and uniform physical properties of crystalline particles, the enhancement of the pharmacokinetics and biodistribution properties of the anticancer drugs are anticipated [142–145].

Nanocrystals can be produced either by top-down or bottom-up methods. The topdown technique involves utilizing a high mechanical energy force to produce nanocrystals from large crystals by media milling or high-pressure homogenization [142,143]. These techniques are generally used to formulate insoluble drugs, especially those used for oral drug delivery [146,147]. In the high-pressure homogenization method, large drug crystals are forced across fluidic pressure and an impact valve, which leads the drug crystals to break down into tinier particles. The control of the particle size is achieved through the pressure and space among the impact valves. On the other hand, in media milling, the grinding of large crystals of the drug is obtained using solid particles like yttrium-stabilized zirconia, cerium, highly crosslinked polystyrene resin-coated beads, and stainless steel [142].

In the bottom-up approach, which involves the antisolvent perception method, nanocrystals can be produced directly from the drug solution. When the drug solution is mixed with an antisolvent with poor drug solubility, in such a case, the decrease in solubility leads to nucleation and crystal growth of the drug, and these are the two critical steps of this method [148]. As more nuclei form during the nucleation stage, then the growth of each nucleus is lower, and based on that, the nucleation step needs to be monitored carefully. The ultrasonic waves produced by sonication can help reduce the size of the nanocrystals by decreasing the particle agglomeration, achieved by breaking down the contact between particles. Consequently, perception and ultrasonication (PU) are commonly used in the bottom-up method [149,150].

The anti-solvent method produced nanocrystals with a smaller size that were costeffective, simpler, and easy to scale up in comparison with other methods of the top-down approach [151,152]. However, various factors during nanocrystal preparation can be controlled to influence the size and morphology of nanocrystals obtained by the antisolvent method, such as the drug concentration, drug solution flow rate, temperature, solvent-toantisolvent volume ratio, stirring speed, and the ultrasound wave characteristics [152–154]. In addition, the addition of surfactants and polymers during the crystallization process has an impact on the size or the shape of the drug's nanocrystals [155]. This shows that engineering the modifications of nanocrystals according to our preference and usefulness is possible. Moreover, a combination of both approaches—the top-down and bottom-up methods—can also possibly obtain NCs with a smaller size (<100 nm), narrow distribution, and less production time [156]. In addition, the shape of nanocrystals is also considered important in controlling the activity and toxicity of anticancer drugs. For instance, the rod shape of some drug nanocrystals has superior anticancer activity and toxicity in comparison with the spherical shape [157]. Another study showed that the needle shape of some drug nanocrystals provides better accumulation in some cancers, which may be referred to as an increasing ability of these nanocrystals to be entrapped [144]. Moreover, the size of the nanocrystals is very critical for the in vivo performance of drugs. For instance, smaller nanocrystals have more dissolution rates than larger ones. Conversely, larger nanocrystals may provide sustained release behavior, which results in greater drug accumulation in tumors similar to drug depots. On the other hand, the smaller one is more stable because of the lower accumulation. Finally, the surface treatment or coating of nanocrystals with a polymer or surfactant can further improve the anticancer properties of the nanocrystals and the stability [10,158].

Manipulation during the preparation of the nanocrystals is possible and might end up in unexpected favorable outcomes. Therefore, this indicates the significance of controlling nanocrystals' properties based on the efficiency, effectiveness, and safety of the anticancer drug, as these can be improved and manipulated indirectly during nanocrystal preparation, especially in the case of the nanocoating.

#### **5. PTX Nanocrystals**

As PTX is a hydrophobic drug with inferior water solubility properties, it is improved greatly by nanocrystal formulation [159]. Based on that, many studies employed the nano-crystallization techniques not only to improve the oral delivery of PTX, but IV, intraperitoneal (IP), and local and intertumoral delivery systems were also developed. Additionally, superior and interesting properties of the PTX NCs were achieved by performing additional modifications to the NCs, such as stabilizing them with surfactants and polymers or coating them with polymers. Tables 1–3 summarize these modified delivery systems by classifying them into three main categories, according to their route of administration: either IV (Table 1), oral (Table 2), or local and intraperitoneal (Table 3) delivery systems. Additionally, the summary tables shed light on the methods used in the preparation of these modified NCs, the in vitro or in vivo models used, and the advantages obtained based on the developed formulations. Nearly the majority of these NCs had a rode-like shape with drug-loading capabilities (>50%), and their size was between <50 and 500 nm (mainly 100–300 nm). The most common route of administration for these novel formulas was the intravenous (IV) route (Table 1), and the most common method of preparation was the antisolvent or precipitation method. The most common cancer cell lines or types of cancer tested were breast cancer (MCF-7 cell lines), followed by ovarian and then lung cancer. Finally, the aims for modifications were mainly focused on providing more solubility and

tumor and cancer cell targeting, less elimination and side effects, and more anti-cancer effects with a smaller dose. In addition, it appears that the NCs' formulation provided a suitable method for multiple drug combinations.

**Table 1.** Modified PTX NC formulations for intravenous (IV) drug delivery.






**Table 2.** Modified PTX NC formulations for oral drug delivery.




#### **Table 3.** Modified PTX NC formulations for local and intraperitoneal drug delivery.


#### **6. Future Aspects**

It is worth mentioning that the nanocrystals are formed by weak, non-covalent interactions. This leads drug nanocrystals to continue to dissolve, albeit slowly, when in contact with water. As such, any surface-coated materials on drug nanocrystals will eventually be detached during the dissolution process. This not only makes it a challenging task to develop surface-treated nanocrystals but also results in transient target-homing effects.

In this regard, the concept of hybrid nanocrystals may overcome this limitation by physically integrating guest molecules among the crystal lattices of nanocrystals. Small molecules such as fluorescent dyes have been demonstrated in vitro and in vivo of paclitaxel nanocrystals. It is thus possible to utilize larger molecules as a guest in making hybrid nanocrystals.

Finally, it is pertinent to understand and eventually predict drug release and dissolution kinetics of paclitaxel nanocrystals in a biological environment. This may be aided by in vitro experimentation and physics-based simulation. One ultimate goal in developing paclitaxel nanocrystals is precision medicine for cancer treatment, which can only be enabled by a thorough understanding of the interactions and the pharmacokinetic characteristics of drug nanocrystals in tissues and cells.

#### **7. Conclusions and Remarks**

Several delivery systems for paclitaxel drugs have been developed to enhance their solubility and pharmacological properties. Of these delivery systems, nanocrystal formulations are considered a promising modality that can also have the advantage of providing a suitable platform for surface modifications. Based on that, many studies employed nano-crystallization techniques not only to improve the oral delivery of PTX but also to improve the IV, intraperitoneal (IP), and local and intertumoral delivery systems, where the applications of surface modifications can be of greater value in terms of targeted delivery.

Moreover, these systems can provide 100% loading and releasing capacities for the drugs as well as gain the advantages of being formulated as particles that have different circulation patterns, fates, cellular uptake mechanisms, and sometimes preferable efficacy and safety profiles compared with free drugs. Finally, more studies are needed to understand the molecular basis for the formation and interaction of these nanocrystals with biological systems, and consequently providing better platforms for useful modifications in the future.

**Author Contributions:** Conceptualization, R.H. and N.A.; methodology, R.H. and N.A.; software, R.H.; validation, R.H., N.A., B.A. and T.L.; formal analysis, R.H. and N.A.; investigation, R.H.; resources, R.H.; data curation, R.H., N.A., B.A. and T.L.; writing—original draft preparation, R.H.; writing—review and editing, N.A. and T.L.; visualization, N.A.; supervision, N.A., B.A. and T.L.; project administration, N.A. and B.A.; funding acquisition, N.A. and B.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Jordan University of Science and Technology, grant number [298/2021].

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Development of Polymer-Based Nanoformulations for Glioblastoma Brain Cancer Therapy and Diagnosis: An Update**

**Bijuli Rabha 1,†, Kaushik Kumar Bharadwaj 1,† , Siddhartha Pati 2,3,† , Bhabesh Kumar Choudhury 4 , Tanmay Sarkar 5,6 , Zulhisyam Abdul Kari 7 , Hisham Atan Edinur 8 , Debabrat Baishya 1, \* and Leonard Ionut Atanase 9, \***


**Abstract:** Brain cancers, mainly high-grade gliomas/glioblastoma, are characterized by uncontrolled proliferation and recurrence with an extremely poor prognosis. Despite various conventional treatment strategies, viz., resection, chemotherapy, and radiotherapy, the outcomes are still inefficient against glioblastoma. The blood–brain barrier is one of the major issues that affect the effective delivery of drugs to the brain for glioblastoma therapy. Various studies have been undergone in order to find novel therapeutic strategies for effective glioblastoma treatment. The advent of nanodiagnostics, i.e., imaging combined with therapies termed as nanotheranostics, can improve the therapeutic efficacy by determining the extent of tumour distribution prior to surgery as well as the response to a treatment regimen after surgery. Polymer nanoparticles gain tremendous attention due to their versatile nature for modification that allows precise targeting, diagnosis, and drug delivery to the brain with minimal adverse side effects. This review addresses the advancements of polymer nanoparticles in drug delivery, diagnosis, and therapy against brain cancer. The mechanisms of drug delivery to the brain of these systems and their future directions are also briefly discussed.

**Keywords:** polymer nanoparticles; glioma/glioblastoma; blood–brain barrier (BBB)/blood brain tumour barrier (BBTB); nanodiagnostics; drug delivery and imaging

#### **1. Introduction**

Cancer is one of the serious life-threatening diseases worldwide with a higher risk of mortality, around 10 million new cases are diagnosed every year [1,2]. Among different types of cancer, brain cancer is the most lethal and invasive type of central nervous system (CNS) disorder [3]. Brain cancer is characterised as a heterogeneous group of primary and metastatic cancers in the CNS [4,5]. The average incidence of both malignant and nonmalignant brain cancer is reported approximately 28.57 per 100,000 population, mostly affecting 0 to 19 years, with a mean annual morbidity rate of 5.57 per 100,000 population [6,7]. Among these, the malignant primary brain cancers with a 5-year survival rate of less than 33.3–35% and even the rate are still alleviating. The average survival span is still not

**Citation:** Rabha, B.; Bharadwaj, K.K.; Pati, S.; Choudhury, B.K.; Sarkar, T.; Kari, Z.A.; Edinur, H.A.; Baishya, D.; Atanase, L.I. Development of Polymer-Based Nanoformulations for Glioblastoma Brain Cancer Therapy and Diagnosis: An Update. *Polymers* **2021**, *13*, 4114. https://doi.org/ 10.3390/polym13234114

Academic Editor: Ki Hyun Bae

Received: 31 October 2021 Accepted: 24 November 2021 Published: 26 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

improved and even lower between 15 to 22 months [8,9]. A recent report from 2020 of the Central Brain Tumor Registry of the United States accounted for primary malignant tumour incidence rate to be 7.08 per 100,000, with 123,484 estimated cases, and 16.71 per 100,000, with 291,927 cases of non-malignant tumour [10]. Malignant primary tumours, i.e., gliomas derived from the glial origin, are newly diagnosed for approximately 70%, mostly in adults [5,11]. The reduced efficacy of brain cancer therapy is mainly attributed to the presence of the blood–brain barrier (BBB) that limits the permeation of systemically applied drugs into the brain [3].

Brain cancers are categorised into two groups, viz., primary brain cancer originated from the brain and resided within the brain, commonly called glioma, and secondary or metastatic brain cancer spreading from primary cancer outside the CNS, originate from systemic neoplasms and further evolved in the interior of brain parenchyma [12,13]. Glial cell originated gliomas include glioblastomas, astrocytomas, schwannomas, oligodendrogliomas, etc. [14]. According to World Health Organization (WHO), glioma tumours of CNS is classified into four grades based on aggressiveness, Grade I pilocytic astrocytoma, Grade II diffuse astrocytoma, Grade III anaplastic astrocytoma, and Grade IV glioblastoma [12]. Glioblastoma (GBM) and its variants were categorised as Grade IV tumours [15]. Grades I and II are considered low-grade glioma, and Grades III and IV are considered high-grade gliomas, i.e., malignant gliomas, and are characterised by poor prognosis [8,16,17]. GBM can either develop from normal brain cells or evolve from preexisting low-grade astrocytoma [18]. GBM is also termed as glioblastoma multiforme or Grade IV astrocytoma [19]. Excessive penetration and vascular proliferation into brain parenchyma is the indication of aggressive cancer [20].

Conventional glioma therapy includes tumour resection followed by radiotherapy and chemotherapy. Surgical resection is generally considered a standard method for glioblastoma therapy. Yet resection of tumour tissue cannot be entirely removed and hence is limited by the glioblastoma's aggressiveness caused by penetration into surrounding tissue microenvironment and tumour vascularisation [20,21]. Hence, tumour resection is associated with the administration of chemotherapeutic drugs and/or radiation therapy for enhanced efficiency. Radiation therapy can be delivered internally or externally and is regarded as the standard treatment for high-grade gliomas [22]. Chemotherapy drugs such as carmustine (BCNU) can cross the BBB and target glioma cells directly [20]. Further, chemotherapy has undergone some alteration by replacing the use of some alkylating agents, viz., carmustine (BCNU), nimustine (ACNU), and lomustine (CCNU) with temozolomide (TMZ) [23]. Temozolomide is converted to 5-3-(methyl)-1-(triazen-1-yl) imidazole-4-carboxamide, at physiological pH, damages DNA via methylation of the O6 position of guanines, blocks DNA replication and induces tumour cell death. Presently, TMZ, along with surgical resection and radiotherapy, is applied for glioblastoma therapy [17]. Despite that, all the treatment strategies possess some limitations towards survival and thus, the prognosis still remains poor (Table 1).

Although brain cancer resembles to other forms of cancer in the body, the major difference is their intracranial neoplasms, heterogeneity, intricate brain system, and the physiological features of the cranial cavity which restrain the treatment options [10]. Gliomas tend to permeate the surrounding tissue microenvironment, and thereby, it is very difficult to determine the tumour boundaries. This also attributes to several difficulties in conventional therapeutic approaches for a curative outcome. Moreover, the physical and chemical barriers hamper therapeutic drug molecules from reaching tumour locations [11]. The BBB and blood–brain tumour barrier (BBTB) represent the diffusion barrier systems of the brain that regulate the influx of drugs to the brain except owing to certain characteristics [24]. Standard treatments remain ineffective due to poor surgical resection of tumours, mainly the infiltrative ones, poor chemo-therapeutic drug influx to the tumour site, and BBB that restrict them from diffusing toward tumour location [25]. The limitations of radiotherapy also result in incomplete eradication of GBM cells resulting in self-renewal and recurrence [26]. Targeting active anticancer agents to the brain is a challenging task in

the area of drug delivery as BBB prevents the transportation of a drug. Hence, higher doses are needed to attain desired therapeutic efficacy which causes undesirable side effects [27].


**Table 1.** Advantages and limitations of conventional glioblastoma therapy.

#### **2. The Blood–Brain Barrier (BBB)**

One of the main hurdles for the effective systemic treatment of brain cancer is the presence of the BBB. The BBB is a semipermeable membrane barrier between blood capillaries and cellular components of brain tissues that control the movement of ions, nutrients, and cells. The BBB also serves for the dynamic transport of nutrients, peptides, proteins and immune cells between the brain and blood [28]. The BBB consists of endothelial cells, glial cells (pericytes, astrocytes, and neurons) and basement membrane [29] (Figure 1). The endothelial cells line the interior brain capillaries forming the tight junctions that allow small molecules, gases and curb the influx of harmful toxins or pathogens such as bacteria, lipophilic neurotoxins, xenobiotics and hydrophilic substances from the blood to the brain [30]. Due to the presence of pinocytic vesicles, other carriers, transport proteins, and large numbers of mitochondria, hydrophobic and essential molecules such as O2, CO2, glucose, hormones, etc. can infiltrate either by passive diffusion or active transport mechanisms [29]. The presence of several transmembrane proteins characterises the tight junctions between the inter-endothelial cells. These protein complexes are mainly comprised of occludin, claudin, and junctional adhesion molecules. These three specialised proteins interact to develop an intricate, tight barrier that is exclusive to the cerebroendothelial cells [31]. The apical part of the endothelial cell is exposed to the brain's blood capillaries, and the basolateral part is exposed to the cerebrospinal fluid supported by the basement membrane. The basement membrane with 30–40 nm thickness consists of Type IV collagen, fibronectin, laminin, heparin sulfate proteoglycans and other extracellular matrix proteins that completely covers the endothelial cells and limits the movement of the solutes [29,31,32]. Approximately 98% of smaller molecular weight drugs and 100% of larger molecular weight drugs are reported for their inability to cross the intact BBB [33,34]. Under various brain-related pathological conditions, including brain cancers, glioma cells loose the structural integrity and the function of the BBB [35]. BBB is compromised in human glioma cells because of the leaky inter endothelial tight junction and poorly differentiated astrocytes that are unable to release essential components for BBB function [31,36]. In this case, it is termed as blood–brain tumour barrier (BBTB) or blood–tumour barrier (BTB) [14] (Figure 1).

**Figure 1.** Schematic representation of blood–brain Barrier (BBB) and the blood–brain tumour barrier (BBTB).

In low-grade gliomas, the structure and function of the BBTB resemble normal BBB, while in hi-grade glioma, BBB is significantly altered, disrupted. Although the degree of BBB disruption varies from the tumour malignancy, low-grade glioma is still a hurdle to treat due to intact BBB. Despite high-grade gliomas, the structural disruption of their vascular density and integrity is negligible to drug permeability in tumour cells [37,38]. However, BBTB is more permeable than the BBB and allows heterogeneous permeability to drugs and other components. Thus, it is a more challenging task to combat the difficulties of brain cancer [39]. Therefore, along with the existing therapeutic regimen, new approaches are required to combat the BBB. To combat these difficulties, various techniques were developed, which are mostly invasive and cause serious side effects. Nanotechnology, especially use of polymer nanoparticles, helps address the major hurdle of glioma therapy non-invasively. Polymer nanoparticles aid in the targeting and delivery of potent drug molecules to the brain. In this review paper, we will briefly summarise the up-to-date existing therapies and diagnoses in brain cancer gliomas using polymer nanoparticles.

#### **3. Polymer Nanoparticles for Drug Delivery Strategy to Overcome the BBB**

ε The BBB is the main problem in the treatment of brain cancer glioma. The chemotherapeutic drugs are mostly ineffective due to limiting permeability to BBB as it allows to pass only low molecular weight (<500 Da), electrically neutral hydrophobic drugs with lipophilicity at log P 2–3 [11,36,40]. The majority of chemotherapeutic drugs are larger in size, ionic, hydrophilic molecules and thus cannot cross the BBB that is attributed to the requirement of a higher systemic dose that results in severe side effects [11]. To overcome these drawbacks, nanoparticles can be utilised for the controlled and sustained delivery of drugs. Biodegradable polymer nanoparticles are extensively studied systems in cancer drug delivery and therapy. These nanoparticles are also highly stable and can be tuned in order to obtain the desirable characteristics for a passive or an active targeting [41]. Polymer nanoparticles can induce selective toxicity and can load ample anticancer drugs or other molecules. Various biodegradable polymeric drug delivery systems include nanogels or hydrogels, poly(ε-caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA), chitosan [42,43], dendrimers, etc. [44]. Due to versatile tuneable properties, these nanoparticles can open tight junctions of BBB, shield BBB limiting properties of anticancer drugs, release the drug

in a sustainable manner, prolong the systemic circulation, and protect against enzymatic degradation [1,45].

Studies showed that Resveratrol loaded PLGA: D-α-tocopheryl polyethylene glycol 1000 succinate blend nanoparticles (RSV-PLGA-BNPs) displayed significant increasing cytotoxicity and enhanced cell penetration in C6 glioma cells. Haemocompatibility evaluation is one of the critical analyses of interaction between nanoparticles and various blood components that determine any adverse effect upon nanoparticle exposure to blood. The nanoparticles should not cause haemolysis during and after infusions. The haemocompatibility analysis of RSV-PLGA-BNPs revealed safe for i.v. administration. The nanoparticles exhibited prolonged systemic circulation up to 36 h. The nanoparticles also showed higher brain accumulation, suggesting a potential system for the betterment of systemic circulation and plasma half-life with a promising anticancer effect against glioma [1]. In another study, L-carnitine-conjugated PLGA NPs were developed to target glioma cells. These NPs were found to significantly cross the BBB and showed a potential anti-glioma effect [46]. Lactoferrin decorated PEG-PLGA NPs was developed for the delivery of shikonin and the treatment of gliomas [47]. Lactoferrin coating promotes internalisation across the BBB. In vitro and in vivo experiments showed the enhanced nanoparticle uptake and distribution of NPs in the brain with effective treatment of glioblastomas.

#### **4. Polymer Nanoparticles for Anticancer Drug Delivery to the Brain: Mechanism**

Polymer nanoparticles can cross BBB or BBTB either passively or via active endocytosis mechanisms. The unmodified polymer NPs internalise BBB mainly through passive mechanism, the so-called enhanced permeability and retention (EPR) effect, which depends on nanoparticle size. However, the NPS internalised by a passive mechanism have comparatively lower brain uptake than ligand-functionalised polymer NPs [48]. Various strategies have been undertaken to improve the infiltration of NPs into the brain. These strategies involve modification of NPs with certain moieties or components to take benefit of BBB endocytosis pathways for drug delivery. Polymer nanoparticles are able to cross BBB/BBTB through adsorption-mediated transcytosis (AMT), carrier-mediated transport (CMT), and receptor-mediated transcytosis (RMT) [49–52] (Figure 2). The internalisation of polymer nanoparticles crossing BBB/BBTB is summarized in Table 2. Polymer nanoparticles with positively charged can electrostatically interact with a negatively charged luminal surface that is attributed to cross the BBB/BBTB. The cationic polymer nanoparticles can be achieved by various surface modification strategies, either by coating or conjugation of cationic polymer or surfactant to non-ionic or neutral polymer. These modifications of NPs have been shown to utilise the AMT mechanisms to improve brain uptake. For example, a study of cationic bovine serum albumin (CBSA) conjugated with poly (ethylene glycol)–b-poly(lactide) (PEG–PLA) nanoparticles (CBSA–NPs), loaded with 6-coumarin was reported for brain delivery. Results revealed that CBSA–NPs uptake in rat brain capillary endothelial cells (BCECs) was enhanced as compared to control group BSA conjugated with pegylated nanoparticles (BSA–NP) BSA–NPs. Fluorescent microscopy of coronal brain sections displayed increased accumulation of CBSA–NPs than of BSA–NPs [53].

In the CMT mechanism, polymers NPs are designed to deliver drugs in order to take advantage of carrier molecules present in BBB. Polymer NPs are modified or decorated with membrane-penetrating components such as amino acids, peptides, and nutrients capable of transporting cargo across the BBB endothelial cells by utilising systemic transporters. For example, 2-deoxy-D-glucose modified poly (ethylene glycol)-co-poly (trimethylene carbonate) nanoparticles (DGlu-NPs) were studied for targeting the glioma BBB. The internalisation of DGlu-NP on RG-2 rat glioma cells was significantly higher than that of non-modified nanoparticles. This was attributed to the recognition of NPs by GLUT1 leading to enhanced cellular internalisation in glioma cells than in surrounding normal tissue and thus exhibiting promising in vivo anti-glioma activity [54].

**Figure 2.** Various transport mechanisms of polymer NPs across blood–brain barrier (BBB).

Similarly, L-carnitine modified PLGA nanoparticles (LC-PLGA NPs) were designed to utilise the advantage of Na-coupled carnitine transporter 2 (OCTN2) expressions on brain capillary endothelial cells as glioma cells for BBB infiltration and targeting. Results showed increased accumulation of NPs in the BBB endothelial cell line (hCMEC/D3) and the glioma cell line (T98G). This revealed the Na dependent cellular uptake that involves OCTN2 in the NPs internalisation process. Moreover, a higher accumulation of LC-PLGA NPs was also observed in the in vivo mouse model study. Furthermore, loading of drugs Taxol and paclitaxel in the LC-PLGA NPs improved anti-glioma activity in both 2D-cell and 3D-spheroid models [46].

With the RMT mechanism, polymer NPs are decorated/designed with targeting ligands that bind to specific cell surface receptors highly expressed in BBB transport pathways. For example, the Transferrin receptor (TfR) is one of the primary targets for investigating RMT across the BBB because of its high expression on BBB/BBTB endothelium [55]. To evaluate in vivo BBB penetration and targeting efficacy, transferrin modified doxorubicin (DOX) and paclitaxel (PTX) loaded magnetic silica PLGA nanoparticles (MNP-MSN-PLGA-Tf NPs) were developed. The nanoparticles were effectively accumulated in the tumour bearing mice suggesting that Tf facilitates NPs delivery across BBB [56].

**Table 2.** Summary of BBB permeability based on polymer-based nanoparticles.



Abbreviation: Adsorption-mediated transcytosis (AMT), carrier-mediated transport (CMT), and receptor-mediated transcytosis (RMT).

#### **5. Polymer Nanoparticles for Brain Cancer Therapy**

Polymer NPs are solid colloidal particles that can be utilised as carriers in which the therapeutic drugs or other active components are dissolved, entrapped, encapsulated, or adsorbed on the surface of the polymer matrix [69]. The structure of the polymer NPs can range from nanospheres to nanocapsules depending on the preparation procedure. Various polymers such as chitosan, gelatin, sodium alginate, albumin and polylactides (PLA), polyglycolides (PGA), poly(lactide co-glycolides) (PLGA), polyanhydrides, polyorthoesters, polycyanoacrylates, poly(ε-caprolactone), poly(glutamic acid), poly(malic acid), poly(Nvinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), poly(acrylamide), poly(ethylene glycol), poly(methacrylic acid) are mostly used in nanoparticle formation for both passive and ligand- functionalized actively targeted therapy [70]. Based on the nature of drugs to be loaded and their route of administration, different synthesis methods were implemented for the production of polymer NPs that include solvent evaporation, solvent diffusion, nanoprecipitation, emulsification, reverse salting out, nano-capsules nano-precipitation, layer-by-layer (LbL) method, etc. [71–73]. The molecular weight, crystallinity, and stability of polymers and the drug's physicochemical properties can be analysed to develop polymeric NPs for drug administration to the brain. Polymeric NPs have a unique ability to reach the tumour site through an active targeting route [74]. Researchers have developed docetaxel (DOC)-loaded PCL and its derivative poly (ethylene glycol)-block-poly(ε−caprolactone) methyl ether (mePEG-PCL) nanoparticles that were dispersed in a bioadhesive film and the formulation exhibited sustained release of drugs. Docetaxel-loaded nanoparticles induced more significant cytotoxicity than free docetaxel for glioma treatment [75]. The study reveals that glycopeptide-engineered poly(d,l-lactideco-glycolide (PLGA) NPs (g7-NPs) provides in vivo evidence of endocytosis of g7-NPs and transported into the endosomes, which help to cross BBB [76]. Gaudin and co-workers have demonstrated the use of convection-enhanced delivery (CED) of NPs for improved chemotherapeutic drugs to the tumour site. They successfully administered gemicitabine, a nucleoside analogue used for the wide range of solid tumours using squalene-based NPs. The study also revealed that PEGylation of the NPs with PEG dramatically improves the distribution of squalene-gemcitabine NPs in the tumours [77].

Most of the current nanomedicines approved by the FDA for clinical use for solid tumour treatment depend on the EPR effect. The enhanced permeability and retention effect or EPR effect is a feature that allows small sized nanoparticles and other active molecules or drugs to pass due to large pore size through leaky vasculature and accumulate in the tumor location.

The brain endothelial cells and glioblastoma cells generally overexpressed a number of receptors, including the low-density lipoprotein receptor, IL-13 receptor, transferrin receptor (TfR), and nicotine acetylcholine receptor that used as drug delivery targets in the brain [78]. Numerous in vivo studies revealed that polymer NPs could circulate for a longer time and accumulate in the tumour site. It is possible to enhance the retention and accumulation of these useful NPs by decorating NPs with tumour-homing ligands such as peptide, aptamer, polysaccharides, saccharides, antibodies, flic acids, etc. [79]. Recently, pluronic micelles (PEG-PPG-PEG) have evolved as perfect candidates for brain therapy, as they can easily cross the BBB and prove their ability to inhibit drug efflux [17]. For instance, Sun et al. developed TfR-T12 peptide-modified PEG-PLA polymer nanoparticle micelles loaded with paclitaxel (PTX) for glioma therapy. They found that the polymeric micelles (TfR-T12-PMs) could be absorbed by tumour cells, cross across BBB monolayers, and inhibit the proliferation of U87MG cells in vitro. A better antiglioma effect with a prolonged median survival of nude mice-bearing glioma was also observed in comparison with unmodified PMs [80]. This suggests that TfR-T12 peptide-modified micelles can cross the BBB system and target glioma cells. In Tables 3 and 4 are shown various synthetic and natural polymer-based NPs for GBMs therapy or diagnosis.

Recently, much research has been carried out on combined photo-based therapy along with other conventional therapy or imaging for glioblastoma treatment. For example, a novel photoacoustic and photothermal guided semiconducting polymer nanoparticles (SPNs) using poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol) (PEG-b-PPG-b-PEG) and SP were reported. The SPNs displayed efficient cellular internalisation for PAI and PTT toward U87 cells and accumulated in subcutaneous as well as brain tumours upon intravenous injection and induced efficient cell death upon NIR-II light irradiation [81].

The recent updates reveal that conjugated polymer nanoparticles (CPNs) are performing well as photosensitiser (PS) in photodynamic therapy (PDT). This efficiency is achieved by CPNs due to their uniform size, biocompatibility, and outstanding ROS production due to extraordinary photo-physical properties as well as fluorescence emission. It is found that porphyrin doped CPNs can eliminate GBMs through ROS-induced apoptotic damage [82].


**Table 3.** Synthetic polymer-based nanoparticles for brain cancer glioma therapy.


Abbreviation: PLG: poly(lactide-coglycolide), DCF-DA: 2′ ,7′ -dichlorofluorescin-diacetate PBAEs: poly (β-amino ester) s, cRGD: cyclic RGD peptides, PGNRs: PEGylated gold nanorods, PEG: polyethylene glycol, PSMA: prostate-specific membrane antigen, NR: nanorods, PCL-diol: poly (ε-caprolactone diol), PU: polyurethane, ALMWP: activatable low molecular weight protamine.


**Table 4.** Natural polymer-based nanoparticles for brain cancer glioma therapy.


Abbreviation: Den: dendrimer, Angio: angiopep-2, PDT: photodynamic therapy, CDP-NP: cyclodextrin-based nanoparticle, TEB: triphenylamine-4-vinyl- (P-methoxy-benzene), DAPI: 4′ ,6-diamidino-2-phenylindole, SPIONs: superparamagnetic iron oxide nanoparticles.

#### **6. Polymer Nanoparticles in the Diagnosis of Brain Cancer**

Before surgery, a high-resolution image using imaging modalities is required for glioma detection. Owing to the invasiveness of glioma cells, determining the exact tumour boundary by eye is challenging. Proper imaging of a tumour is essential for assessing the extent of tumour distribution before surgery and the response to a treatment regimen after surgery [5]. Several available techniques for visualisation and diagnosis of brain cancer glioma include optical and ultrasound (US) imaging, photoacoustic (PA) imaging, computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and fluorescence (FL) imaging techniques (Figure 3) [112]. Currently, magnetic resonance imaging (MRI), a non-invasive technique that can detect the size, shape, and tumour location, is initially employed diagnostic method for patients with suspected GBM [113]. MRI can determine the boundaries of the tumour tissues and/or intraoperative to elucidate tumour outline during surgical resection by applying gadolinium (Gd). Due to a shorter half-life, Gd must be administered often to maintain blood levels for efficient scanning. The use of intraoperative ultrasonography to obtain integrated brain tissue imaging is another non-optical method. However, this approach

does not provide enough information for detecting smaller or superficial brain tumours. Other invasive techniques for analysing brain tumour tissues include Raman spectroscopy, optical coherence tomography, fluorescence spectroscopy, and thermal imaging [114]. Computed tomography (CT) can also be used to determine the presence of the tumour. Still, its use is relatively lesser in clinics for diagnosing GBM due to poor resolution compared to MRI [115]. Likewise, positron emission tomography (PET) imaging with 11C-methionine could be an effective diagnostic tool for GBM patients' prognosis [116,117]. To understand cancer tumours, precise preoperative imaging and painless sensitive postimaging techniques to provide real-time data are demanded. Current imaging modalities, however, lack accuracy, sensitivity, and specificity. Nanotechnology has sparked interest in bioimaging and biosensing in recent years.

**Figure 3.** Polymer NPs in imaging for improved diagnosis of brain cancer.

'Nanodiagnostics' combined with nanotechnology could provide a drug delivery system with traditional diagnostic and imaging procedures [118,119]. Nanotechnology has made it easier to acquire data with great precision and accuracy while avoiding invasive procedures. NPs with tunable optical, magnetic, and electrical properties are able to provide diagnostic tools for detection and imaging brain cancer/tumours [120]. Biocompatible NPs owing ideal physical characteristics, such as surface chemistry, morphology, solubility, stability, etc., facilitate drug delivery and imaging as it acts as image contrast agents [121]. Polymer NPs could be a good reservoir system for drugs and a platform for additional modification for efficient tumour targeting or imaging [122]. Polymer NPs possess various advantages in drug delivery to the brain that can entrap or carry drugs that prevent them from metabolism and excretion. Moreover, NPs can easily transport drugs across the BBB without changing the barrier properties [31,123,124]. In this section, polymer NPs utilised in the diagnosis and detection of brain cancer glioma until now are primarily focused. The imaging and diagnosis techniques currently being investigated with reference to polymer NPs are listed in Table 5.

Polymer-based superparamagnetic NPs have mainly been employed as drug delivery systems and contrast agents in MRI imaging. These NPs are highly stable and biocompatible, can prolong systemic circulation time, have drug loading ability and control of drug release, and combine with their magnetic performance for MRI [125]. Ganipineni et al. synthesised paclitaxel (PTX) and superparamagnetic iron oxide (SPIO)-loaded PEGylated PLGA-based NPs (PTX/SPIONPs) and analysed for therapeutic efficacy in an orthotopic U87MG model. The cellular internalisation of these NPs was found to be concentration dependent. The MRI scanning displayed the blood–brain barrier disruption in the glioma affected location. Moreover, enhanced accumulation was also observed in ex vivo biodistribution analysis of GBM-bearing mice with magnetic targeting [126]. Researchers have evaluated SPIO-loaded brain penetrating PLGA NPs by CED administration on rat models and visualised using positron emission tomography (PET) and MRI [127]. SPIO-loaded NPs showed excellent transverse (T2) relaxivity. After CED of NPs, the biodistribution in the brain was analysed using MRI, which revealed a period of one month longer signal attenuation of SPIO-loaded brain-penetrating PLGA NPs. The co-administration of SPIO-loaded PLGA NPs allows intraoperative monitoring of biodistribution in the brain in order to ensure the delivery to tumour location and therapeutic effect over time [127]. Researchers have developed Polysorbate 80 coated temozolomide-loaded PLGA-based superparamagnetic nanoparticles (P80- TMZ/SPIO-NPs), evaluated for anti-glioma activity and analysed as a diagnostic agent for MRI [128]. The superparamagnetic P80-TMZ/SPIO-NPs showed a significant antiproliferative effect and remarkable cellular internalisation on C6 glioma cells. Moreover, the in vitro MRI scanning revealed that P80-TMZ/SPIO-NPs could also serve as a good contrast agent [128].



Abbreviation: *N*-(4-[18F] fluorobenzyl) propanamido-PEG4-Biotin, brain cancer stem cells (BCSCs).

#### **7. Limitations and Challenges**

From the past times, tremendous developments have been evidenced in brain cancer therapy. Yet, there have not been emerged significant changes in mortality rate and

improving patients' quality of life. Although nanoparticle-based drug delivery systems have brought a new horizon, many challenges remain and need to be solved in the future. The development of effective polymeric NPs for drug delivery and targeting is a challenging task for clinical translations. The advantage and limitations are summarised in Figure 4. The toxicity of these systems is one of the main challenges. The slow degradation rate of polymer NPs induce a longer circulation time in the body and could cause unknown complications.

Further, extensive investigations are required for optimization of the NPs. One of the major obstacles in clinical translation is the interaction of NPs and biological systems. Upon entering the complicated biological system, the designed polymeric NPs will instantly interact with neighboring biomolecules, leading to the formation of protein corona that alters their properties. This affects NPs size, stability, surface properties and determines the pharmacokinetics, biodistribution, cellular internalisation, intracellular trafficking, immune system, and toxicity [133–136]. In addition, more in vitro and in vivo studies are required to better understand the mechanisms in targeted nanoparticle-based therapy. Several essential factors related to the in vivo behaviour of NPs and their effect on other healthy brain cells are hence required to be extensively examined. Currently, there is still insufficient pre-clinical data of polymer-based NPs on brain delivery, data to correlate in vitro-in vivo observation, which makes it difficult to conclude about their therapeutic efficacy.

**Figure 4.** The advantages and limitations of polymer NPs in drug delivery and therapy.

#### **8. Future Perspective and Conclusions**

Glial originated brain cancers are the most aggressive gliomas that depict a threat to humans. The conventional therapies are still inefficient to overcome due to tumor heterogeneity and, specifically, the blood–brain barrier (BBB) of malignant gliomas. The polymeric nanoparticles-based brain cancer therapy approaches are currently gaining interest due to the drug safety, controllable drug release, and efficient targeting in tumors. Most importantly, reports revealed that polymer NPs could even transport across BBB. In this review article, we summarize the newest breakthroughs in the use of polymer nanocarriers for drug delivery, therapy and diagnosis of brain cancer are explored, emphasizing how

they are a critical aspect of modern anticancer drug delivery strategies. Various polymer NPs have been generated to reduce anticancer drug losses, premature degradation, enhance drug availability, and reduce drug toxicity by improving drug accumulation in specific organs and tissues. Although the potential impact of polymer NPs in cancer therapy is exceedingly promising, numerous obstacles that currently limit their widespread clinical usage must be solved. For polymer NPs to be used in clinical trials, long-term safety investigations must be conducted in various animal models to eliminate the possibility of non-endogenous components accumulating in the body causing any harm. As a result, huge costs must be provided when conducting in vivo pharmacokinetic studies to evaluate the applicability in the human body. Another factor to consider is the challenges that may arise when transitioning from laboratory to large-scale production. The scaling up of the preparatory process is a major obstacle that must be surmounted. A significant number of polymer NPs are currently in the pre-clinical stage of development, but only one system has entered a clinical study. This is primarily because several challenges impede further development, such as a lack of potency in animal models and toxicity concerns. To overcome the aforementioned concerns, researchers need to focus more on new therapeutic innovations such as revising fabrication processes to modify and improve polymeric NPs in order to accommodate the demand for various anticancer drugs for effective clinical feasibility. New therapeutic innovations also include novel therapeutic strategies for combination therapy and stimuli-activated drug delivery. For example, delivering two or more anticancer drugs simultaneously might enhance the treatment of various cancer developments by targeting different tumour related signalling pathways, resulting in a synergistic therapeutic impact. In addition, the researcher needs to improve the targeting of cancer stem cells (CSCs) for effective cancer therapeutic effect as CSCs is a critical factor for tumour recurrence. In conclusion, pre-clinical experimentation and clinical trials are mandatory for an efficient polymer nanoparticle-based anticancer therapy. Hopefully, all of these developments will lead to more patient-specific and targeted anticancer therapies.

**Author Contributions:** Conceptualization, D.B., K.K.B. and B.R.; methodology, K.K.B., B.R., T.S., and S.P.; formal analysis, K.K.B., B.R., T.S., B.K.C. and D.B.; investigation S.P., T.S., K.K.B. and L.I.A.; writing—original draft preparation, K.K.B., B.R., T.S., S.P., B.K.C., D.B., Z.A.K., H.A.E. writing review and editing, K.K.B., B.R., T.S., S.P., B.K.C., D.B. and L.I.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by a research grant from the University Grants Commission (NFST) vide Grant No. F1-17.1/2015-16/NFST-2015-17-ST-ASS-3863.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The study did not report any data.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

