2.5.4. Proton Nuclear Magnetic Resonance (1H NMR)

2.5.3. FT-IR Spectral Analysis FT-IR spectroscopy characterized the chemical stability of NPs encapsulated in the core of the biopolymer. The FT-IR spectra of Erlo, biopolymer, polyvinyl alcohol, Dox– Erlo NPs, and Dox–Erlo-NP conjugates are indicated in Figure 6. The structure of Erlo shows a 2-methoxy ethoxy group (C-O stretching) and amino-group (N-H stretching) of The formation of an amide bond between the primary amine group (-NH2) of folic acid and the carboxylic acid group from the Dox–Erlo NPs through a conjugation reaction is shown in Figure 7A. The <sup>1</sup>H NMR spectroscopy of the amide linkage formation in Dox– Erlo-NP conjugates is shown in Figure 7A,B. An appearance of the signals at 8.3921 ppm indicated the formation of an amide bond through a reaction between the activated ester group of the polymeric nanoparticles and the primary amine group of folic acid (Figure 7B).

#### quinazoline ring. The biopolymer demonstrated a peak around 2743.12 cm−1, and 2918.33 2.5.5. X-ray Diffraction Analysis

cm−1 belongs to the carboxylic acid group. The Erlo drug demonstrated absorption bands at 3267.14 cm−1, corresponding to N-H stretching, and at 1081.18 cm−1, attributed to C-O stretching (Figure 6). The confirmation of the physicochemical drug behavior encapsulation in the NPs was further illustrated with help of X-ray analysis. The X-ray diffraction patterns of Erlo, cinnamon biopolymer, Dox–Erlo NPs, and Dox–Erlo-NP conjugates are shown in Figure 8. The high-intensity characteristic peaks in the Erlo were observed at 2θ angles of 18.74◦ , 20.38◦ , 21.07◦ , 25.26◦ , 36.04◦ , and 40.33◦ ,indicating their crystalline nature (Figure 8A).The low-intensity peaks in the biopolymer were observed at 2θ angles of18.84◦ , 22.72◦ , 23.48◦ , and 25.41◦ . Moreover, the peaks prevailed in the biopolymer, as is shown in Figure 8B, suggesting a less-crystalline nature. However, the peaks of crystalline nature were produced at a very low intensity or disappeared in the diffraction patterns of Dox–Erlo NPs and the Dox–Erlo conjugates, indicating that Erlo and Dox were in amorphous or molecular states in the NPs.

#### 2.5.6. In Vitro Drug Release

In vitro release studies were performed for Erlo and Dox from Dox–Erlo NPs and Dox–Erlo-NP conjugates at a pH of 7.4 (simulating a physiological pH) and a pH of 5.4 (mimicking the pH of acidic, intracellular, endosomal cancer cells), respectively. The maximum amounts of Dox released from the Dox–Erlo NPs and Dox–Erlo-NP conjugates at a pH of 7.4 were 59.54 ± 0.10% and 58.34 ± 0.073%, respectively. On the other hand, at

a pH of 5.4, the maximum amounts of Dox release from the Dox–Erlo NPs and Dox–Erlo-NP conjugates were 76.29 ± 0.19% and 74.24 ± 0.24%, respectively. The amounts of Erlo released from Dox–Erlo NPs and Dox–Erlo-NP conjugates at a pH of 7.4 were 70.42 ± 0.05% and 68.47 ± 0.29%, respectively. Similarly, at a pH of 5.4, the amounts of Erlo released at the end of 47 h were 82.11 ± 0.30% and 78.43 ± 0.39%, respectively, as is shown in Figure 9A,B. *Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 12 of 30

**Figure 6.** The FT–IR Spectra of Erlo (**A**), cinnamon polymer (**B**), polyvinyl alcohol (**C**), Dox–Erlo NPs (**D**), and Dox–Erlo NP conjugates (**E**). **Figure 6.** The FT–IR Spectra of Erlo (**A**), cinnamon polymer (**B**), polyvinyl alcohol (**C**), Dox–Erlo NPs (**D**), and Dox–Erlo NP conjugates (**E**).

2.5.4. Proton Nuclear Magnetic Resonance (1H NMR)

The formation of an amide bond between the primary amine group (-NH2) of folic

acid and the carboxylic acid group from the Dox–Erlo NPs through a conjugation reaction is shown in Figure 7A. The 1H NMR spectroscopy of the amide linkage formation in Dox– Erlo-NP conjugates is shown in Figure 7A,B. An appearance of the signals at 8.3921 ppm indicated the formation of an amide bond through a reaction between the activated ester

**Figure 7.** Schematic diagram of amide bond formation of Dox–Erlo NPs (**A**) and the1H NMR spectrum of Dox–Erlo-NP conjugates (**B**). **Figure 7.** Schematic diagram of amide bond formation of Dox–Erlo NPs (**A**) and the1H NMR spectrum of Dox–Erlo-NP conjugates (**B**).

The confirmation of the physicochemical drug behavior encapsulation in the NPs

was further illustrated with help of X-ray analysis. The X-ray diffraction patterns of Erlo, cinnamon biopolymer, Dox–Erlo NPs, and Dox–Erlo-NP conjugates are shown in Figure 8. The high-intensity characteristic peaks in the Erlo were observed at 2θ angles of 18.74°,

2.5.5. X-ray Diffraction Analysis

2.5.6. In Vitro Drug Release

20.38°, 21.07°, 25.26°, 36.04°, and 40.33°,indicating their crystalline nature (Figure 8A).The low-intensity peaks in the biopolymer were observed at 2θ angles of18.84°, 22.72°, 23.48°, and 25.41°. Moreover, the peaks prevailed in the biopolymer, as is shown in Figure 8B, suggesting a less-crystalline nature. However, the peaks of crystalline nature were produced at a very low intensity or disappeared in the diffraction patterns of Dox–Erlo NPs

**Figure 8.** The XRD diffraction analyses of Erlo (**A**), cinnamon biopolymer (**B**), Dox–Erlo NPs (**C**), and Dox–Erlo-NP conjugates (**D**). **Figure 8.** The XRD diffraction analyses of Erlo (**A**), cinnamon biopolymer (**B**), Dox–Erlo NPs (**C**), and Dox–Erlo-NP conjugates (**D**).

In vitro release studies were performed for Erlo and Dox from Dox–Erlo NPs and

Dox–Erlo-NP conjugates at a pH of 7.4 (simulating a physiological pH) and a pH of 5.4 (mimicking the pH of acidic, intracellular, endosomal cancer cells), respectively. The maximum amounts of Dox released from the Dox–Erlo NPs and Dox–Erlo-NP conjugates at a pH of 7.4 were 59.54 ± 0.10% and 58.34 ± 0.073%, respectively. On the other hand, at a pH of 5.4, the maximum amounts of Dox release from the Dox–Erlo NPs and Dox–Erlo-NP

leased from Dox–Erlo NPs and Dox–Erlo-NP conjugates at a pH of 7.4 were 70.42 ± 0.05% and 68.47 ± 0.29%, respectively. Similarly, at a pH of 5.4, the amounts of Erlo released at

the end of 47 h were 82.11 ± 0.30% and 78.43 ± 0.39%, respectively, as is shown in Figure

**Figure 9.** In vitro drug releases of Erlo and Dox from the plain drug, Dox–Erlo NPs, and Dox–Erlo-**Figure 9.** In vitro drug releases of Erlo and Dox from the plain drug, Dox–Erlo NPs, and Dox–Erlo-NP conjugates at a pH of 5.4 (**A**) and a pH of 7.4 (**B**).

NP conjugates at a pH of 5.4 (**A**) and a pH of 7.4 (**B**).

#### 2.5.7. Kinetic Release Model

The releases of Erlo and Dox from Dox–Erlo NPs and their conjugates were fitted to the different release kinetic model. The exponent (*n*) expressed aFickian or non-Fickian pattern of drug release. The exponent value for the zero-order release/Case II transport, *n* = 1; non-Fickian diffusion, 0.5 < *n* < 1; or relaxational release, *n* > 1 is considered. The model of good fit was judged based on the regression coefficient value (R<sup>2</sup> ). The regression coefficient (R<sup>2</sup> ) values for such models were determined, for example: zero order (R<sup>2</sup> = 0.9002), first order (R<sup>2</sup> = 0.9744), Higuchi (R<sup>2</sup> = 0.9096), Korsmeyer–Peppas (R<sup>2</sup> = 0.9793), and Hixson–Crowell (R<sup>2</sup> = 0.9593) were estimated. It was observed that Korsmeyer–Peppas showed a good fit to the model of (R<sup>2</sup> = 0.9793) for the Erlo release from Dox–Erlo-NP conjugates at apH of5.4. The *n*-value was 0.4805 and the k-value was 3.0543. The release of Dox from Dox–Erlo-NP conjugates at a pH of 5.4 fitted in a different kinetic model, and the regression coefficients for various kinetic models were provided: zero order (R<sup>2</sup> = 0.8541), First order (R<sup>2</sup> = 0.9231), Higuchi matrix (R<sup>2</sup> = 0.9233), Korsmeyer–Peppas (R<sup>2</sup> = 0.9751), and Hixson–Crowell (R<sup>2</sup> =0.9025) [Table 6]. The Korsmeyer–Peppas was the best-fitted model, with a regression value of (R<sup>2</sup> = 0.9751), an *n*-value of 0.5951, and a k-value of 2.4551. The release mechanism indicated an anomalous, non-Fickian diffusion of Dox, both via diffusion and biopolymeric matrix erosion; on the other hand, Erlo was released via biopolymeric matrix erosion.

**Table 6.** Kinetic drug release of Erlo and Dox release from Dox–Erlo-NP conjugates at pH 5.4.


In the determination of the Erlo release from the Dox–Erlo-NP conjugates at a pH of 7.4, the regression coefficient values for the zero order (R<sup>2</sup> = 0.8704), first order (R<sup>2</sup> = 0.9537), Higuchi matrix (R<sup>2</sup> = 0.9190), Korsmeyer–Peppas (R<sup>2</sup> = 0.9782), and Hixson–Crowell (R<sup>2</sup> = 0.9306) were determined. Among these models, Korsmeyer–Peppas demonstrated the highest regression value (R<sup>2</sup> = 0.9782), with a release exponent *n*-value of 0.5294 and a k-value of 2.733 selected. Further, the regression coefficients for Dox release from Dox–Erlo-NP conjugates at a pH of 7.4 were determined using the same models: zero order (R<sup>2</sup> = 0.8718), first order (R<sup>2</sup> = 0.9294), Higuchi (R<sup>2</sup> = 0.9062), Korsmeyer–Peppas (R<sup>2</sup> = 0.9709), and Hixson–Crowell (R<sup>2</sup> =0.9125). Due to the emergence of a highest regression coefficient value for the Korsmeyer–Peppas model, it was selected as the model of good fit. It indicated an *n*-value of 0.41 and a k-value of 2.9451 [Table 7]. The mechanism of drug release expressed that Dox was released via Fickian diffusion following both diffusion and biopolymeric matrix erosion. On the other hand, the Erlo release mechanism followed an anomalous or non-Fickian diffusion through biopolymeric matrix erosion [27].


**Table 7.** Kinetic drug release of Erlo and Dox release from Dox–Erlo-NP conjugates at pH 7.4.

#### 2.5.8. Hemolysis Study

Hemolysis experiments were carried out to ensure the biocompatibility of the inhouse-built NPs and NP conjugates in the bloodstream and to obtain information about the charge–particle interaction with biomolecules in terms of thrombosis and hemolysis in vivo. These interactions enable damage to erythrocytes and thereby acquit hemoglobin from erythrocytes. It was observed that increasing the NP doses led to an increased release of hemoglobin from the erythrocytes. The hemolytic analysis revealed that RBC damages were less than 6–8% in any of the concentrations (1.5 mg, 3 mg, and 6 mg) used in the experiment relating to placebo NPs, Dox–Erlo NPs, and Dox–Erlo-NP conjugates.

#### 2.5.9. Cytotoxicity Assay

The results of the MTT assay analysis of plain drugs, Dox–Erlo-NPs, and Dox–Erlo-NP conjugates on glioma cell lines (U87 and C6) at varying concentrations (0.20 µM, 0.40 µM, 0.80 µM, 1.6 µM, 3.2 µM, and 6.4 µM) are shown in Figure 10A,B. The Dox– Erlo-NP conjugates significantly depleted the count of viable cells to 24.66 ± 2.08% when compared to Dox–ErloNPs (66 ± 2.6%) and plain drugs (85.33 ± 5.5%) in glioma U87 cells. Oppositely, Dox–Erlo-NP conjugates reduced the viable cell count to 32.33 ± 2.51% when compared to Dox–ErloNPs (65 ± 1%) and plain drugs (87 ± 3.46%) in glioma C6 cells. Furthermore, cell death was expressed in terms of the IC50 related to the dose of drug, which killed 50% of cancer cells in a specified time period, i.e., the inhibitory concentration (IC50). The IC50 values of plain Dox–Erlo, Dox–ErloNPs, and Dox–Erlo-NP conjugates were 26.589 µM, 9.830 µM, and 3.064 µM, respectively, after 24 h in the U87 cell line. The IC50 values of plain Dox–Erlo, Dox–ErloNPs, and Dox–Erlo-NP conjugates were determined to be 32.60 µM, 8.625 µM, and 3.350 µM, respectively, after 24 h in the C6 glioma cell line, shown in Figure 10A,B.

#### 2.5.10. Biodistribution Study

The tissue homogenates from various organs such as the liver, kidney, brain, and blood of rats were extracted and analyzed via HPLC for the presence of Dox and Erlo. It was found that a significant amount of Dox and Erlo were estimated in the brain as compared to drug suspension (*p* < 0.05). The biodistribution studies of the formulation in various organs are expressed in Figure 11.

Dox–ErloNPs (65 ± 1%) and plain drugs (87 ± 3.46%) in glioma C6 cells. Furthermore, cell death was expressed in terms of the IC50 related to the dose of drug, which killed 50% of cancer cells in a specified time period, i.e., the inhibitory concentration (IC50). The IC50 values of plain Dox–Erlo, Dox–ErloNPs, and Dox–Erlo-NP conjugates were 26.589 µM, 9.830 µM, and 3.064 µM, respectively, after 24 h in the U87 cell line. The IC50 values of plain Dox–Erlo, Dox–ErloNPs, and Dox–Erlo-NP conjugates were determined to be 32.60

**Figure 10.** The percentage cell viability following 24 h of treatment with various doses of plain Dox– Erlo, Dox–ErloNPs, Dox–Erlo-NP conjugates, placebo Dox–Erlo NPs, and placebo Dox–Erlo-NP conjugates on U87 (**A**) and C6 (**B**) glioma cell lines. The experiments were performed in triplicate with mean ± S.D (*n* = 3). Significance value \* (*p <* 0.05), \*\* (*p <* 0.01), \*\*\* (*p <* 0.001), \*\*\*\* (*p <* 0.0001) **Figure 10.** The percentage cell viability following 24 h of treatment with various doses of plain Dox–Erlo, Dox–ErloNPs, Dox–Erlo-NP conjugates, placebo Dox–Erlo NPs, and placebo Dox–Erlo-NP conjugates on U87 (**A**) and C6 (**B**) glioma cell lines. The experiments were performed in triplicate with mean ± S.D (*n* = 3). Significance value \* (*p <* 0.05), \*\* (*p <* 0.01), \*\*\* (*p <* 0.001), \*\*\*\* (*p <* 0.0001) relative to pure Dox-Erlo.

relative to pure Dox-Erlo.

2.5.10. Biodistribution Study

**Drug concentration (ng/gm of tissue)**

The tissue homogenates from various organs such as the liver, kidney, brain, and blood of rats were extracted and analyzed via HPLC for the presence of Dox and Erlo. It was found that a significant amount of Dox and Erlo were estimated in the brain as compared to drug suspension (*p* < 0.05). The biodistribution studies of the formulation in var-

**Figure 11.** Graph representing the biodistribution of Dox (**A**) and Erlo (**B**) from plain, Dox–Erlo NPs, and Dox–Erlo-NP conjugates. Isolated organs of animals after 24 h of dose (**C**).

## 2.5.11. Stability Study

The stability study was performed as per guideline issues under a stability study [28]. The Dox–Erlo-NP conjugates' stability experiments under a specific set of conditions are expressed in Table 8. The particle size observed was 109.45 ± 12.48 nm at 25 ± 2 ◦C, 65 ± 5% RH at the end of 90 days. However, at an elevated temperature of 40 ± 2 ◦C, 75 ± 5% RH, a particle size of 115.33 ± 12.38 nm was observed. Similarly, the surface charge on the Dox–Erlo-NP conjugates at temperatures of 25 ± 2 ◦C, 65 ± 5% RH and 40 ± 2 ◦C, 75 ± 5% RH were recorded as −21.1 ± 4.01 and −20.4 ± 3.20 mV, respectively. The entrapment efficiencies after a stability period of 90 days were calculated at 76 ± 5.3% and 73 ± 3.3%, respectively.

**Table 8.** Stability indicating data of Dox–Erlo-NP conjugates with regard to particle size, zeta potential, and % entrapment efficiency.


#### **3. Discussion**

The treatment of a glioma is impeded via the invasiveness or the inadequacy of drugs penetrating the BBB [29]. The current study was designed to develop, characterize, and evaluate Dox–Erlo NPs and folate-armored Dox–Erlo-NP conjugates for targeting glioma cancer via a nose-to-brain route. The study aimed to improve the targeted specificity and promote the penetration of NPs to glioma cells to achieve the desired therapeutic concentration.

This biopolymeric, nanocarrier-based drug delivery is a novel approach for drug targeting to a specific region as it offers biodegradability and biocompatibility and is nontoxic to the vital organs of the body, as was disclosed in the hemocompatibility study. The folate-armored, polymeric nanocarrier has shown better biodistribution in the brain due to its higher permeability and penetration of the BBB. The developed biopolymer nanoconjugates were effective in glioma therapy as they enabled a controlled drug release over a prolonged time and a tunable size, by which they could approach the target domain, minimize off-target effects, and increase bio-stability. The TEM studies of the nanoconjugate were well-dispersed, uniform, de-aggregated, and consistent in size. The low PDI value showed that the developed preparations were consistent, homogeneous, and had a narrow size distribution. The zeta potential value indicated a negative surface charge on the nanoparticle formulation; the nanoparticles showed no agglomeration due to a samecharge surface repellence of each other, creating a resistive force that led to the enhanced stability of the nanosize system [30].

It has been proven that the over-expressed folate receptor on the tumor cells' surface could be a specific target site for delivering cytotoxic agents [31]. In our study, the conjugation of folic acid to Erlo–Dox preparations was found to be at a higher concentration in the brain when compared to a non-conjugated preparation. This substantiates the higher efficacy of folate-conjugated nanoparticles when compared to plain NPs. The conjugated NPs' formulation exhibited a remarkable cell death and higher concentration in the brain when compared to the unconjugated NPs, consistent with the previous literature. The conjugation of folate with NPs was confirmed by <sup>1</sup>H NMR analysis. The results clearly

indicated that conjugated NPs can be a promising, tumor-targeting carrier candidate. No endothermic peak in DSC was detected for the drug in Dox–Erlo NPs and conjugated NPs, suggesting that drug has been incorporated in the NPs. The DSC chromatogram of mannitol was detected in the Dox–Erlo-NP conjugates [32].

The functional peaks of the drug in FT-IR becoming flattened in the Dox–Erlo NPs and Dox–Erlo-NP conjugates indicated that the drug was encapsulated in the biopolymeric core [33]. <sup>1</sup>H NMR evidently revealed the conjugation of the primary amine group of folic acid with the carboxylic acid group of the polymeric NPs. In <sup>1</sup>H NMR, the appearance of the signals at 8.3921 ppm indicated the formation of an amide bond by a reaction between the activated ester group of the polymeric nanoparticles and the primary amine group of the folic acid. Dox–Gefit-NP conjugates were synthesized, as indicated by the formation of amide bond. The appearance of this peak confirmed the conjugation of folic acid [34].

The prepared nanoparticles were nano-sized, having a desirable diameter of 95.35 ± 10.25 nm and 110.12 ± 9.2 nm for the NPs and conjugates, respectively, and exhibited a sustained release of the drug under physiological conditions [35,36].The zeta potential value indicated a negative surface charge on the nanoparticle formulation and no non-agglomerated NPs, probably due to the same-charge surface repellence of each other, with the resultant resistive force leading to an enhanced stability of the nanosize system [37]. Furthermore, the mean PDI of the NPs in our study was 0.109, showing that the developed preparations were consistent, homogeneous, had a narrow particle-size distribution, were monodispersed, and were satisfactory [38,39].

Free Dox and Erlo can cause brain toxicity, cardiotoxicity, and kidney or liver damage. In this study, converting them to NPs and encapsulating them within a biopolymer helped to prevent the toxic side effects of systemic Dox and Erlo administration. The encapsulation of the drug was confirmed by DSC, with FTIR analysis as standard practice. It is being proven that biopolymers demonstrate non-toxicity and short immunogenicity, are bioabsorbable, and have subsequently good biocompatibility. Hence, their use can minimize the potential hazards of cytotoxicity. In the present study, a *Cinnamomum zeylanicum* biopolymer was extracted and used as a nanoparticle-carrier material to achieve a higher concentration of the drug at the targeted tumor site with reduced toxicity. The cytotoxicity study result showed no toxicity of the biopolymer, signifying that the biopolymer is safe and biocompatible [40].

The results of the % drug release assessment demonstrated that Erlo released faster than Dox from NPs. During the initial phase of drug release, an abrupt release was demonstrated, followed a controlled release for a long time. This may be due to Erlo becoming entrapped in the exterior layer, while Dox was encapsulated in the interior core of the NPs [41]. Further, it was observed that release of Erlo and Dox was found to be higher at an acidic, intracellular, endosomal pH of 5.4 when compared to a pH of 7.4. It is worthwhile to disclose herein that the microenvironment of a tumor is slightly more acidic than the physiological fluid [42]. The higher drug release at an endosomal pH of 5.4 in the slightly acidic microenvironment of the tumor may be attributed to the fact that the protonation of the biopolymer and drugs resulted in a higher dissolution of Dox and Erlo from the internal polymeric complex of the NPs in the acidic environment. The pH-dependent drug release is highly desirable for cancer-tissue targeting and also minimizes non-selective drug release in systemic circulation. It also provides sufficient drug concentration upon cellular internalization, which is mediated via endosomal escape and lysosomal fusion [43–45]. After fitting the drug-release data in kinetic models, the exponent value *n* of Erlo from Dox–Erlo NPs at a pH of 5.4 and a pH of 7.4 and Dox from Dox–Erlo NPs at a pH of 5.4 showed that the release mechanism was diffusion (non-Fickian). However, Dox at a pH of 7.4 demonstrated a Fickian drug release mechanism. The findings indicate that Erlo and Dox release from Dox–Erlo-NP conjugates was ascertained via diffusion from polymeric core. The hemolysis assay disclosed that the maximum concentration of formulation was 6 mg; when tested for hemocompatibility, this did not cause significant hemolysis. The hemolysis study was resembled preceding work in the

literature [46,47]. The developed formulations were conceived to be as least toxic or nontoxic and are regarded as safe and hemocompatible for in vivo administration. As per the experimental observation, the stability of the Dox–Erlo-NP conjugates were maintained, as indicated by an insignificant alteration in particle size, zeta potential, and entrapment efficiency after an analysis of the sample at fixed intervals of time during a storage period of 90 days (*p* > 0.05). This further indicates that in-house-built Dox–Erlo NPs were robust and consistently in line with the ICH stability-testing guidelines [48,49].

The efficacy of the formulation was studied by assessing the IC50 values and the percent of depletion of cancer cells [50]. The cell-killing potency of the formulations was dose and time-dependent. The MTT assay interpreted that the Dox–Erlo-NP conjugates successfully decreased the % cell viability according to the concentration of the drug in NPs and the drug delivery into the cells [51]. The existing literature demonstrates that using a synergistic combination of EGFR inhibitor viz., Erlotinib provides the cells susceptible to apoptosis with exposure to the DNA-destructive agent doxorubicin [52].

The analytical estimation showed that drug concentration was achieved in the vital organs (the heart, liver, and kidney) with small quantities of Dox and Erlo when compared to the targeted brain, which may be attributed to the partitioning behavior of the nanosized Dox–Erlo NPs and the Dox–Erlo-NP conjugates via endothelial fenestration. Overall, the concentrations of Dox and Erlo achieved in the target organ, i.e., in the brain, were significantly higher than in other organs of the body (*p* < 0.05), indicating the specific delivery of the formulated conjugate in the targeted region of glioma cancer [53].

#### **4. Material and Methods**

#### *4.1. Materials*

Erlotinib (Mol wt = 393.436, purity of ≥95%) was a gift sample from Natco Pharma Ltd. UPSIDC (Dehradun, India). Doxorubicin also a gift sample from Neon Laboratories Pvt. Ltd. (Ghaziabad, India).The cinnamon biopolymer was purchased from Shree Ram Overseas (New Delhi, India). The polyvinyl alcohol (PVA) was received from Sisco Research Laboratory Pvt. Ltd. (Mumbai, India). The cross-linking agents EDC [1-(3 Dimethylaminopropyl)-3-Ethyl Carbodiimide Hydrochloride] and Sulpho-NHS [N-Hydroxysuccinimide] were received from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). The solvent, Dimethyl Sulfoxide (DMSO), was obtained from Merck Pvt. Ltd. (Mumbai, India), and acetone was obtained from SD Fine Chem Pvt. Ltd. (Mumbai, India), HPLC-grade water and other reagents were used as received.

#### *4.2. Cytotoxicity Study*

#### Materials

The specified materials for the study of cytotoxicity, such as culture media, penicillin streptomycin, MTT (4, five-dimethylthiazol-2yl)-2, five-diphenyl tetrazolium bromide), fetal bovine serum (FBS), and Dulbecco's Modified Eagle Medium (DMEM) were bought from Himedia (Mumbai, India). The phosphate-buffered saline (PBS) was purchased from (Himedia, India). The cell lines C6 and U87 were received from NCCS, Pune, India. Cells were stored at 37 ◦C and 5% CO<sup>2</sup> in a humidified CO<sup>2</sup> incubator to maintain continuous growth.

#### *4.3. Formulation Optimization Using Statistical Design*

The optimization of formulation was carried out through Design-Expert Software (Design-Expert version 12, State-Ease® Inc., Minneapolis, MN, USA) using Box–Behnken design (BBD).The expert design used a three-level, three-factor BBD which produced seventeen experimental runs for optimizing the formulation. The investigative impact of independent variables, viz., (A) polymer concentration; (B) surfactant concentration; and (C) sonication time on thefactors (R1) particle size (nm); (R2) PDI, and (R3) drug release (%) were studied. The levels of independent variables under study were used as low (−1), intermediate (0), and high (+1), and their impact on the responses R1, R2, and

R3 are shown in Table 1. This design comprehensively explained the major, combined, and quadratic effect of factors A, B, and C on various selected responses in the study of the formulation. The optimization of formulation based on Design-Expert version 12, State-Ease® Inc. (Minneapolis, MN, USA) was reported in various preceding works [54]. ratic effect of factors A, B, and C on various selected responses in the study of the formulation. The optimization of formulation based on Design-Expert version 12, State-Ease® Inc. (Minneapolis, MN, USA) was reported in various preceding works [54].

independent variables, viz., (A) polymer concentration; (B) surfactant concentration; and (C) sonication time on thefactors (R1) particle size (nm); (R2) PDI, and (R3) drug release (%) were studied. The levels of independent variables under study were used as low (−1), intermediate (0), and high (+1), and their impact on the responses R1, R2, and R3 are shown in Table 1. This design comprehensively explained the major, combined, and quad-

#### *4.4. Preparation of Dox–Erlo-Loaded NPs 4.4. Preparation of Dox–Erlo-Loaded NPs*

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 23 of 30

Dox–Erlo-loaded biopolymeric NPs were developed by implementing a modified, double-emulsion solvent-evaporation technique [55]. The technique involved the preparation of an Erlo solution in an organic phase (1 mg/mL), a Dox solution in an aqueous phase (5 µg/mL), a biopolymer in an aqueous phase (29.4 mg/mL), and the preparation of an aqueous PVA solution. Primarily, the solutions of Erlo (1 mg/mL) and Dox (5 µg/mL) were transferred slowly using an injectable needle in the aqueous biopolymer solution (2.94% *w*/*v*) and emulsified slowly using a probe sonicator (Hielscher ultrasonicator, Berlin, Germany) (02 min, 30 KHz power, 50 W, 01 cycle) to obtain a polymeric core of the drug as a primary emulsion (*o/w*). Second, this primary emulsion was transferred into the aqueous PVA solution (2.20 % *w*/*v*) slowly, using an injection needle at a rate of 0.5 mL/min. This was then emulsified for 11 min with the probe sonicator (30 KHz power, 80 W, 01 cycle) to obtain a secondary emulsion comprising a nanoparticle suspension. Thereafter, the preparation was stirred magnetically at 1000× *g* rpm for 4 h at ambient temperature to allow for the evaporation of the organic phase. Further, NPs were held open overnight to obtain hard and dry particles. The nanoparticles were then ultracentrifuged at 15,000× *g* rpm (OptimaTM LE-80K Ultracentrifuge) for 30 min and washed (*n* = 3) to free the NPs of un-entrapped drug and free biopolymer matter. The Dox–Erlo-loaded nanoparticle was then re-dispersed in water and lyophilized to dryness for future characterization. The NP preparation steps are illustrated in Figure 12. Dox–Erlo-loaded biopolymeric NPs were developed by implementing a modified, double-emulsion solvent-evaporation technique [55]. The technique involved the preparation of an Erlo solution in an organic phase (1 mg/mL), a Dox solution in an aqueous phase (5 µg/mL), a biopolymer in an aqueous phase (29.4 mg/mL), and the preparation of an aqueous PVA solution. Primarily, the solutions of Erlo (1 mg/mL) and Dox (5µg/mL) were transferred slowly using an injectable needle in the aqueous biopolymer solution (2.94% *w/v*) and emulsified slowly using a probe sonicator (Hielscher ultrasonicator, Berlin, Germany) (02 min, 30 KHz power, 50 W, 01 cycle) to obtain a polymeric core of the drug as a primary emulsion (*o/w*). Second, this primary emulsion was transferred into the aqueous PVA solution (2.20 % *w/v*) slowly, using an injection needle at a rate of 0.5 mL/min. This was then emulsified for 11 min with the probe sonicator (30 KHz power, 80W, 01 cycle) to obtain a secondary emulsion comprising a nanoparticle suspension. Thereafter, the preparation was stirred magnetically at 1000× *g* rpm for 4 h at ambient temperature to allow for the evaporation of the organic phase. Further, NPs were held open overnight to obtain hard and dry particles. The nanoparticles were then ultracentrifuged at 15,000× *g* rpm (OptimaTM LE-80K Ultracentrifuge) for 30 min and washed (*n* = 3) to free the NPs of un-entrapped drug and free biopolymer matter. The Dox–Erlo-loaded nanoparticle was then re-dispersed in water and lyophilized to dryness for future characterization. The NP preparation steps are illustrated in Figure 12.

**Figure 12.** Schematic representation of the preparation of Dox–Erlo NPs by double-emulsion evap-**Figure 12.** Schematic representation of the preparation of Dox–Erlo NPs by double-emulsion evaporation method.

#### *4.5. Surface Modification of Dox–Erlo Biopolymeric NPs*

oration method.

The nanoparticles were re-dispersed to 10 mg/mLin double-distilled water and incubated with 0.1% of 1-(3 Dimethylaminopropyl)-3-Ethyl Carbodiimide Hydrochloride (EDC. HCl) and N-hydroxysuccinimide (sulpho-NHS, 0.05% *w*/*v*), for 5 h in a biological shaker to activate the carboxylic group. In the course of the first step of the coupling reaction, an unstable intermediate was formed on reaction with the EDC cross-linker, which fur-

ther reacted with sulpho-NHS and formed a stable ester. After incubation, the aminereactive stable ester (sulpho-NHS NPs) was washed three times with distilled water. In the consequent step, the stable ester (sulpho-NHS NPs) was re-dispersed with folic acid (0.1% *w*/*v*) and incubated overnight to hasten the coupling reaction at ambient temperature in an end-to-end biological shaker. The folate-conjugated Dox–Erlo NPs were subjected to centrifugation for half an hour at 15,000× *g* rpm; thereafter, the supernatant was withdrawn and washed to remove traces of un-conjugated EDC and sulpho-NHS. The conjugated Dox–Erlo NPs were dried via lyophilization for further use. The surface-modification steps of the NPs are shown in Figure 13. further reacted with sulpho-NHS and formed a stable ester. After incubation,the aminereactive stable ester (sulpho-NHS NPs) was washed three times with distilled water. In the consequent step, the stable ester (sulpho-NHS NPs) was re-dispersed with folic acid (0.1% *w/v*) and incubated overnight to hasten the coupling reaction at ambient temperature in an end-to-end biological shaker. The folate-conjugated Dox–Erlo NPs were subjected to centrifugation for half an hour at 15,000× *g* rpm; thereafter, the supernatant was withdrawn and washed to remove traces of un-conjugated EDC and sulpho-NHS. The conjugated Dox–Erlo NPs were dried via lyophilization for further use. The surface-modification steps of the NPs are shown in Figure 13.

The nanoparticles were re-dispersed to 10 mg/mLin double-distilled water and incubated with 0.1% of 1-(3 Dimethylaminopropyl)-3-Ethyl Carbodiimide Hydrochloride (EDC. HCl) and N-hydroxysuccinimide (sulpho-NHS, 0.05% *w/v*), for 5 h in a biological shaker to activate the carboxylic group. In the course of the first step of the coupling reaction, an unstable intermediate was formed on reaction with the EDC cross-linker, which

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 24 of 30

*4.5. Surface Modification of Dox–Erlo Biopolymeric NPs* 

**Figure 13.** Surface modification of Dox–Erlo NPs. **Figure 13.** Surface modification of Dox–Erlo NPs.

#### *4.6. Characterization of Dox–ErloNanoparticles 4.6. Characterization of Dox–ErloNanoparticles*

#### 4.6.1. Particle Analysis and Z-Average

4.6.1. Particle Analysis and Z-Average The distribution of particles and the Z-average of Dox–Erlo NPs were analyzed by utilizing a Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK). As per the standard procedure, the Dox–Erlo NPs were re-distributed in HPLC-grade water (0.5 mg/mL) and sonicated for one minute for one cycle at 60 Hz. The sizing analyses were The distribution of particles and the Z-average of Dox–Erlo NPs were analyzed by utilizing a Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK). As per the standard procedure, the Dox–Erlo NPs were re-distributed in HPLC-grade water (0.5 mg/mL) and sonicated for one minute for one cycle at 60 Hz. The sizing analyses were computed and recorded three times (*n* = 3).
