**1. Introduction**

**\***

In the last decades, grea<sup>t</sup> attention has been paid to the development of nanoscale vehicles for drug delivery [1–3]. The incorporation of drug molecules into nanocarriers allows us to overcome poor water solubility of hydrophobic drugs, as well as increase stability against hydrolytic degradation of hydrophilic ones [4]. Moreover, nanoparticulate drug formulations can act in a targeted and prolonged manner, enhancing the efficacy of treatment, e.g., cancer treatment [4]. Platinum-based complexes (cisplatin, carboplatin, oxaliplatin, etc.) are widely used chemotherapeutics agents for the treatment of various types of cancer [5,6]. Cisplatin (*cis*-(eblock)dichloridoplatinum(II)) is the first-generation platinum drug, that has a therapeutic effect against breast cancer, ovarian cancer, lung and head and neck cancer, cervix carcinoma, etc. However, it produces significant side effects such as ototoxicity, hematological, and emetogenicity [7]. Carboplatin (cis-diammine(1,1- cyclobutanedicarboxylato)platinum(II)), that is the second-generation platinum complex and cisplatin analogue, was designed to reduce the dose limiting toxicity of cisplatin. Oxaliplatin ((*trans*-R,R-cyclohexane-1,2-diamine)oxalatoplatinum(II)) is the third-generation platinum complex that was designed to overcome cellular resistance to cisplatin and carboplatin. Oxaliplatin shows higher solubility and less toxicity than cisplatin. It is used as a standard

**Citation:** Kadina, Y.A.; Razuvaeva, E.V.; Streltsov, D.R.; Sedush, N.G.; Shtykova, E.V.; Kulebyakina, A.I.; Puchkov, A.A.; Volkov, D.S.; Nazarov, A.A.; Chvalun, S.N. Poly(Ethylene Glycol)-*b*-Poly(D,L-Lactide) Nanoparticles as Potential Carriers for Anticancer Drug Oxaliplatin. *Molecules* **2021**, *26*, 602. https:// doi.org/10.3390/molecules26030602

Academic Editors: Marek Brzezi ´nski and Małgorzata Ba´sko

Received: 23 December 2020 Accepted: 21 January 2021 Published: 24 January 2021

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**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/).

7

treatment for colorectal cancer. Moreover, oxaliplatin can be active against refractory ovarian cancer, germ-cell cancers, non-small cell lung cancer, etc. Nevertheless, its low water solubility, short half-life in the bloodstream, and non-selective biodistribution reduces the effective dose of oxaliplatin in the targeted tissues and enhances the systemic toxicity [8].

Design of nanocarriers for oxaliplatin delivery is one of the strategies to overcome its limitations and improve the efficacy of cancer treatment [7–9]. A wide range of various types of nanocarriers including inorganic nanoparticles [10], dendrimers [11], liposomes [12], polymeric nanoparticles [13], block-copolymer micelles [14,15], nanogels [16], etc., have been investigated for drug delivery of oxaliplatin. Several liposomal formulations of oxaliplatin, e.g., Lipoxal (Regulon, Inc.) and MBP-426 (Mebiopharm Co., Ltd.), are under clinical investigation [12]. Lipoxal is based on oxaliplatin-loaded PEGylated liposomes prepared from soy phosphatidylcholine, cholesterol, dipalmitoyl phosphatidylglycerol, and mPEG-distearoyl phosphatidylethanolamine, which exhibits reduced side effects compared to free oxaliplatin [17]. MBP-426 is a transferrin (Tf)-conjugated N-glutaryl phosphatidylethanolamine liposomal formulation of oxaliplatin, which can provide selective tumor targeting by binding to transferrin receptors [18]. Nonetheless, this technology has some disadvantages, namely rapid leakage of the incorporated drug molecules from liposomes during storage and after administration [19]. The promising nanoformulations of oxaliplatin are based on various polymeric carriers. The strategies of oxaliplatin incorporation into polymeric vehicles are generally based on chemical conjugation (complexation) or physical encapsulation. Chemical conjugation is usually accomplished between block copolymers, containing poly(glutamic acid) Pglu [20–22], poly(methacrylic acid) (PMA) [14], or poly(lactide-*co*-2-methyl-2-carboxyl-propylene carbonate) P(LA-*co*-MCC) [23,24] block and active part of oxaliplatin dichloro(1,2-diaminocyclohexane) platinum(II) DACHPt. Cabral et al. prepared DACHPt-loaded poly(ethylene glycol)-*b*-Pglu PEG-*b*-Pglu micelles, consisting of Pglu core surrounded by PEG corona, through polymermetal complex formation of DACHPt with Pglu block [20,21]. It was observed that an increase of the initial [DACHPt]/[Glu] molar ratio from 0.25 to 1.5 leads to enhancement of platinum encapsulation efficiency from 20% to 90% and a growth of hydrodynamic diameter of DACHPt-loaded micelles from 25 to 50 nm [20]. In ref. [21], the effect of PEG272-*b*-Pglu*n* (*n* = 20, 40, 70) copolymer composition on the DACHPt loading efficacy and biodistribution in vivo of DACHPt-loaded micelles was studied. It was observed that incorporation efficacy of DACHPt was approximately 30%, regardless of Pglu block length (molar ratio [DACHPt]/[Pglu] = 1). Meanwhile, in vivo biodistribution assay performed on tumor-bearing mice showed that DACHPt-loaded micelles based on PEG272-*b*-Pglu20 copolymer with the shortest Pglu block exhibited the lowest non-specific accumulation in normal tissues (liver, kidney, spleen) providing the highest accumulation in tumor. The authors suggested that PEG272-*b*-Pglu20 copolymer allows the formation of particles with effective surface coverage by PEG leading to reduction of accumulation of particles in liver. Although chemical conjugation usually leads to higher drug loading content in micelles, polymers can coordinate with Pt-complexes in a non-specific geometry, resulting in uncontrolled crosslinking [25]. Physical encapsulation of oxaliplatin into polymeric carriers allows us to overcome this limitation. Cui et al. developed poly(lactide) PLA nanoparticles stabilized by Tween80 as potential carriers for oxaliplatin [13]. The authors studied the effect of formulation parameters on the size, stability, drug loading content, and encapsulation efficacy of the nanoparticles. Optimal oxaliplatin loading content and its encapsulation efficacy was found to be 3.52 ± 0.07 wt.% and 17.40 ± 0.47 %, correspondingly. Micelles based on a stearic acid-grafted chitosan oligosaccharide CSO-SA were also investigated as carriers for oxaliplatin [26]. The highest drug loading content in CSO-SA micelles was 3.5 wt.%, and its encapsulation efficiency was 47%. The authors reported that a chitosan-based nanoformulation of oxaliplatin demonstrates enhanced antitumor activity in vitro against several cancer cells (about 3–6 folds) compared to free oxaliplatin. Despite a number of nanoformulations developed using different platforms, none of them have received FDA approval at the moment. The design of new types of nanocarriers for

delivery of oxaliplatin is of high interest. We believe that PLA-*b*-PEG nanoparticles are a promising platform due to their flexibility and successful track record as nanocarriers for development of targeted anticancer drug formulations.

Nanoparticles of amphiphilic block copolymers comprising PLA hydrophobic block and PEG hydrophilic block have been extensively studied as potential vehicles for drug delivery due to their biocompatibility and biodegradability [27–29]. In aqueous solution, PLA-*b*-PEG copolymers are able to self-organize into "core-corona" nanosized structures, where hydrophobic PLA block chains form an inner core surrounded by a corona of hydrophilic PEG block chains [27,28]. PEG chains provide a "stealth effect" for the nanoparticles, minimize their undesirable interaction with proteins and capturing by the reticuloendothelial system, that extends circulation time of the nanoparticles in the body [4]. The nanoscale size of these block copolymer particles is favorable for passive targeting due to the so-called enhanced permeability and retention (EPR) effect. It is known that tumors and inflamed tissues are characterized by increased vascular permeability and impaired lymphatic drainage, which result in selective accumulation of nanoparticles into them [30]. Depending on the characteristics of the block copolymer (composition, molecular architecture, and block lengths) as well as the preparation conditions, PLA-*b*-PEG nanoparticles of different sizes and morphologies can be obtained [30]. Garofalo et al. showed that PLA/PEG block copolymer architecture, as well as its chemical composition, strongly affect size, stability, and tumor uptake of the micelles [27]. Linear and "tree-shaped" block copolymers mPEG45-*b*-(PLA)*n* and mPEG113-*b*-(PLA)*<sup>n</sup>*, where *n* = 1 and 2 or 4, with various length and tacticity of the hydrophobic PLA blocks, were synthesized. It was reported that only the two-arm mPEG45-*b*-poly(D,L-lactide)2 mPEG45-*b*-(P(D,L)LA)2 copolymer produces high-stable monodispersed micelles with a hydrodynamic diameter of about 250 nm, whereas the other block copolymers show low stability and tendency to aggregate with formation of large submicron clusters. The influence of hydrophobic block length on the size of P(D,L)LA-*b*-PEG nanoparticles was studied in ref. [31]. It was reported that the hydrodynamic diameter of P(D,L)LA-*b*-PEG113 nanoparticles produced by nanoprecipitation increased from 27.7 to 174.6 nm, with an increase of P(D,L)LA block molecular weight from 3 to 110 kDa. The effect of the nanoprecipitation parameters on the size of mPEG113-*b*-P(D,L)LA800 nanoparticles was studied by Y. Dong and S.-S. Feng [32]. The increase of the polymer concentration in organic phase from 4 to 13 g/L leads to increasing hydrodynamic diameter of the particles from 77 to 111.2 nm. Meanwhile, the increase of the organic solvent volume from 5 to 25 mL with a fixed polymer concentration of 10 g/L results in reducing hydrodynamic diameter of the particles from 89.8 to 79.7 nm.

For application of block copolymer nanoparticles as carriers for drug delivery, considerable attention should be given to their structure. Small-angle X-ray (SAXS) and neutron (SANS) scattering are powerful techniques for characterization of nanostructure, which can be implemented to analyze micelles and nanoparticles in aqueous medium [33]. Important characteristics affecting physicochemical properties of core-corona nanoparticles, and their drug loading capability can be determined from small-angle scattering data, e.g., density and size of the core, thickness and surface density of the corona, etc. [34–38]. Using SANS, T. Riley et al. studied the structure of deuterated P(D,L)LA(d)-*b*-PEG nanoparticles in aqueous solution, prepared by solvent evaporation method [36]. It was found that the P(D,L)LA(d) block length affects the core size as well as conformation of the corona-forming PEG chains. Thus, P(D,L)LA(d)-*b*-PEG113 with short P(D,L)LA block (3 kDa) forms nanoparticles with a small core and highly splayed PEG chains in corona. The increase of the P(D,L)LA molecular weight to 15 kDa at a fixed length of the PEG block leads to increasing core size and, consequently, reducing PEG chain grafting density. The authors also observed that with decreasing PEG chain grafting density, the corona becomes more radially homogeneous. Using SAXS, Ma et al. investigated the influence of PLA stereostructure on the density of core and doxorubicin loading efficacy of PLA/PEG nanoparticles, which were prepared by a precipitation/solvent evaporation method [37]. It was observed that nanoparticles formed of enantiomerically-mixed P(L)LA64-*b*-PEG113/P(D)LA71-*b*-PEG113

copolymers and P(L)LA64-*b*-PEG113 copolymer exhibit larger core density compared with nanoparticles with stereoblock copolymer core, e.g., PEG113-*b*-P(L)LA32-P(D)LA34, and P(D,L)LA58-*b*-PEG113.

Various fabrication techniques were developed to incorporate drugs in the core of the nanoparticles or at the core-corona interface, e.g., direct dissolution, dialysis, nanoprecipitation, salting-out method, emulsification method, etc. [39]. There are many factors that affect drug loading, including drug-core compatibility [40–42], hydrophobic block length [31,43–45], its crystallization capability [37,44,45], preparation conditions [32], etc. Despite the recent progress in development of the drug nanoformulations based on nanoparticles of amphiphilic block copolymers, there is still a lack of systematic data on the relationship between the chemical structure of block copolymer and the drug loading ability of nanoparticles. X. Zhang et al. investigated nanoparticles of P(D,L)LA-*b*-mPEG copolymers with various P(D,L)LA/mPEG weight ratios prepared by a solution casting method as potential carriers of highly hydrophobic drug paclitaxel [43]. It was reported that the particles based on P(D,L)LA-*b*-mPEG copolymers with higher P(D,L)LA content exhibited enhanced paclitaxel loading efficacy. Nanoparticles prepared by a nanoprecipitation method from P(D,L)LA-*b*-PEG copolymers with a fixed molecular weight of PEG block (5 kDa) and a variable molecular weight of P(D,L)LA block (from 3 to 110 kDa) were studied as a delivery system for watersoluble drug procaine hydrochloride [31]. It was demonstrated that the drug incorporation efficacy was independent of the P(D,L)LA block molecular weight.

PEG-*b*-PLA nanoparticles demonstrate many advantages as carriers for anticancer drugs. However, to the best of our knowledge, there is only one research article dedicated to PEG-*b*-PLA nanoparticles as potential carriers of oxaliplatin [15]. In addition, there is no data on the relationship between the chemical structure of PEG-*b*-PLA copolymers and the oxaliplatin loading ability of these nanoparticles. Development of such nanoformulation is complicated due to hydrophilicity of oxaliplatin, which limits its loading into carriers. The precise control of nanoparticles' structure and properties throughout adjustment of molecular structure of amphiphilic PEG-*b*-PLA copolymers is a promising approach for tuning their loading capacity. In the present work, we propose a nanoformulation of Pt(II)-based complex oxaliplatin based on mPEG113-*b*-P(D,L)LA*n* nanoparticles, where *n* = 62–173 monomer units. Amphiphilic block copolymers with rather short PLA blocks (< 200 monomer units) are of grea<sup>t</sup> interest because significant changes in structure and characteristics of nanoparticles can occur with increasing PLA block length in this range of polymerization degrees. The aim of our study was to elucidate the effect of molecular weight of the hydrophobic P(D,L)LA block on the size, structure, morphology, and drug loading of the mPEG-*b*-P(D,L)LA nanoparticles.

## **2. Results and Discussion**

## *2.1. Synthesis of mPEG-b-P(D,L)LA Copolymers*

Amphiphilic mPEG113-*b*-P(D,L)LA*n* copolymers with fixed molecular weight of the PEG block were synthesized by ring-opening polymerization (Figure 1). It is known that hydroxyl-containing compounds act as co-initiators in a coordination-insertion polymerization of lactide in presence of SnOct2 catalyst [46]. Therefore, block-copolymers can be synthesized with mPEG as a macroinitiator.

Molecular characteristics of the synthesized polymers are presented in Table 1. The residual content of monomer determined by 1H NMR was less than 1% for all block copolymers.

**Figure 1.** Scheme of synthesis of mPEG-*b*-P(D,L)LA copolymers by ring-opening polymerization.


**Table 1.** Molecular characteristics of the synthesized diblock copolymers

1 Determined by 1H NMR. 2 Determined by GPC.

## *2.2. Characterization of Drug-Free mPEG-b-(D,L)LA Nanoparticles*

For targeted delivery through EPR-effect hydrodynamic diameter of nanoparticles should be in the range of 10–200 nm [30]. Therefore, physicochemical characteristics of nanoparticles are important parameters that should be studied in order to consider them as potential drug carriers. Intensity size distribution curves (DLS) of aqueous suspensions of drug-free mPEG113-*b*-P(D,L)LA*n* nanoparticles are presented in Figure 2a. Monomodal distribution with hydrodynamic radius *Rh* values corresponding to the peak maximum of 22 ± 10 and 28 ± 10 nm are observed for mPEG113-*b*-P(D,L)LA135 and mPEG113-*b*-P(D,L)LA173, respectively. A second peak appears on the intensity size distribution curve of mPEG113-*b*-P(D,L)LA62 nanoparticles with the shortest PLA block. One can sugges<sup>t</sup> that these two peaks can be attributed to small individual nanoparticles and their large aggregates. For individual mPEG113-*b*-P(D,L)LA62 nanoparticles and their aggregates the values of *Rh* corresponding to the peak position were found to be 16 ± 6 and 72 ± 40 nm, respectively. Since the light scattering intensity of large objects is much stronger than that of small objects [47] and the peak intensities are almost equal, one can assume that only a minor fraction of the aggregates co-exists with the main fraction of individual nanoparticles. This assumption was also confirmed by TEM and SAXS measurements, provided below.

**Figure 2.** (**a**) DLS intensity size distribution curves for nanoparticles based on mPEG113-*b*-P(D,L)LA*n* (c = 0.5 g/L): 1— mPEG113-*b*-P(D,L)LA173, 2—mPEG113-*b*-P(D,L)LA135, 3—mPEG113-*b*-P(D,L)LA62; (**b**) representative TEM image of spherical individual mPEG113-*b*-P(D,L)LA62 nanoparticles.

ζ-Potential of drug-free mPEG113-*b*-P(D,L)LA*n* nanoparticles was found to be in the range from −14 to −20 mV (Table 2), which promotes their high stability in water. ForallthemPEG113-*b*-P(D,L)LA*n*copolymers,TEMrevealsonlysphericalindividual

 nanoparticles (Figure 2b).

In order to evaluate the size and address the structure of nanoparticles, SAXS measurements were carried out. Scattering curves for aqueous suspensions of mPEG113-*b*-P(D,L)LA*n* nanoparticles in Log *I*–Log *s* coordinates are presented in Figure 3a. All the SAXS curves converge to a plateau in the region of *s* < 0.1 nm<sup>−</sup>1, indicating the absence of large scattering objects (aggregates) in aqueous suspensions of mPEG113-*b*-P(D,L)LA*n*

nanoparticles [48]. Apparently, the aggregates were removed during centrifugation before SAXS measurements. One also can see the secondary maximum at the SAXS curves in the region 0.3 < *s* < 1 nm<sup>−</sup><sup>1</sup> (Figure 3a), suggesting that the nanoparticles have a spherical, well-defined structure with a relatively narrow size distribution.

**Sample** *Rh***1, nm** *Rg***2, nm** *Rg***3, nm** *Dmax/2***4, nm** *R***5, nm** *2Rg/Dmax Rg/R* ζ **6, mV** mPEG113-*b*-P(D,L)LA173 28 ± 10 11.4 ± 0.1 12.5 ± 0.1 19.5 ± 1 15.4 ± 1 0.64 ± 0.05 0.81 ± 0.06 −14 ± 8 mPEG113-*b*-P(D,L)LA135 22 ± 10 10.1 ± 0.1 9.7 ± 0.1 17 ± 1 11.5 ± 1 0.57 ± 0.06 0.84 ± 0.09 −15 ± 4 mPEG113-*b*-P(D,L)LA6216 ± 6 9.1 ± 0.1 8.6 ± 0.1 14 ± 1 9 ± 1 0.61 ± 0.07 0.96 ± 0.11 −20 ± 4

**Table 2.** Characteristics of the mPEG113-*b*-P(D,L)LA*n* nanoparticles in aqueous suspensions.

1 The value of *Rh* corresponding to the peak position on DLS intensity size distribution curve. 2 Gyration radius of nanoparticles calculated from Guinier plots. 3 Gyration radius of nanoparticles evaluated from *P(R)*. 4 Radius of nanoparticles evaluated from *P(R)*. 5 The value of *R* corresponding to the maximum position on *P(R)* function. 6 ζ-potential of nanoparticles.

**Figure 3.** (**a**) SAXS (small-angle X-ray) curves in Log *I*–Log *s* coordinates and (**b**) corresponding pair distance distribution functions *P(R)*–*R* for mPEG113-*b*-P(D,L)LA*n* nanoparticles: 1—mPEG113-*b*-P(D,L)LA173 (c = 7.5 g/L), 2—mPEG113-*b*-P(D,L)LA135 (c = 5 g/L), 3—mPEG113-*b*-P(D,L)LA62 (c = 5 g/L). The SAXS curves are shifted vertically for clarity.

The bell-shaped Kratky plots indicate a compact globular shape of the nanoparticles (Figure 4b) [49].

The values of gyration radius *Rg* were determined from the Guinier plots (Figure S1 of the Supplementary Materials). It was found that an increase in the P(D,L)LA block length results in higher *Rg* values (Table 2).

The pair distance distribution functions *P(R)* are presented in Figure 3b. They are bellshaped with a peak shifted to distances smaller than a half of maximum dimension of the scattering objects *Dmax/2* (Figure 3b). Since solid spherical particles display bell-shaped *P(R)* functions with a peak at about *Dmax/2* [50], one can assume that the shift of *P(R)* function maximum could be attributed to "core-corona" structure of the mPEG113-*b*-P(D,L)LA*n* nanoparticles.

**Figure 4.** (**a**) SAXS curves in Log *I*–Log *s* coordinates for oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles: 1— mPEG113-*b*-P(D,L)LA173 (c = 7.5 g/L), 2—mPEG113-*b*-P(D,L)LA135 (c = 5 g/L), 3—mPEG113-*b*-P(D,L)LA62 (c = 5 g/L). The SAXS curves are shifted vertically for clarity. (**b**) Kratky plots for oxaliplatin-loaded (1, 2, 3) and drug-free (1a, 2a, 3a) mPEG113-*b*-P(D,L)LA*n* nanoparticles: 1—mPEG113-*b*-P(D,L)LA173 (c = 7.5 g/L), 2—mPEG113-*b*-P(D,L)LA135 (c = 5 g/L), 3,—mPEG113-*b*-P(D,L)LA62 (c = 5 g/L).

The values of *R* and *Dmax/2* corresponding to the peak position and the maximum dimension evaluated from *P(R)* functions are listed in Table 2. It was found that an increase in the P(D,L)LA block length leads to higher *R* and *Dmax/2* values. *Rg* values evaluated from *P(R)* functions were compared with *Dmax/2* and *R* values. The *2Rg/Dmax* values were found to be in the range of 0.57–0.64, whereas *Rg/R* was in the range of 0.81–0.96 (Table 2). It should be noted that for spherical particles with constant density *Rg/R* = 0.78. Thus, one can suppose that the mPEG113-*b*-P(D,L)LA*n* nanoparticles have higher electron density in the inner part than that in the outer part, i.e., a "core-corona" structure [37,48,51]. It should be noted that the PLA block makes the main contribution to the X-ray scattering due to its higher electron density in comparison with less dense PEG corona. The values of *Dmax/2* and *R* are smaller than the values of *Rh* or evaluated by DLS (Table 2), which can be explained by higher contribution of the corona.

One can see in Table 2 that the radius of nanoparticles determined both by DSL and SAXS increases with an increase in length of amorphous P(D,L)LA block, which is in accordance with the literature [31,52,53]. Enhanced hydrophobic interactions result in higher aggregation number of the copolymer chains into a nanoparticle leading to an increase in hydrodynamic size [52]. Variation of the polymerization degree of P(D,L)LA block allows us to effectively tune the size of nanoparticles that could be important in optimizing the drug incorporation into these potential drug delivery carriers.

According to the literature the size of obtained mPEG113-*b*-P(D,L)LA*n* nanoparticles, which is less than 100 nm, could provide delivery of the loaded anticancer agen<sup>t</sup> to tumor in passive targeting manner [30], achieve high tumor extravasation efficacy, and deep tumor penetration of particles regardless of the tumor type [54].

## *2.3. Characterization of Oxaliplatin-Loaded mPEG-b-(D,L)LA Nanoparticles*

The values of oxaliplatin loading content in mPEG113-*b*-P(D,L)LA*n* nanoparticles after removal of free drug are listed in Table 3, which demonstrates that the length of hydrophobic PLA block in mPEG113-*b*-P(D,L)LA*n* copolymers affects the drug loading. The highest loading content of oxaliplatin was evaluated as 3.8 wt.% for nanoparticles of mPEG113-*b*-P(D,L)LA62 copolymer with the shortest PLA block. Due to low hydrophobicity of oxaliplatin, its incorporation in the hydrophobic PLA core is unfavorable [12]. We suppose that the anticancer agen<sup>t</sup> could be adsorbed at the core-corona interface of the mPEG113-*b*-P(D,L)LA*n* nanoparticles due to the highest density of hydrophilic PEG chains

there. The size of the nanoparticles decreases with a decrease of PLA block length (Table 3). The smaller size of the mPEG113-*b*-P(D,L)LA*n* nanoparticles results in enhanced core-corona interface leading to an increase in oxaliplatin loading content.

**Table 3.** Characteristics of oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles in aqueous suspensions.


1 The value of *Rh* corresponding to the peak position on DLS intensity size distribution curve. 2 Gyration radius of nanoparticles calculated from Guinier plots. 3 Gyration radius of nanoparticles evaluated from *P(R)*. 4 Radius of nanoparticles evaluated from *P(R)*. 5 The value of *R* corresponding to the maximum position on *P(R)* function. 6 ζ-potential of nanoparticles. 7 Drug loading content determined by ICP-AES.

> In order to investigate the influence of oxaliplatin loading on size, morphology, and structure of the mPEG113-*b*-P(D,L)LA*n* nanoparticles, DLS, TEM, and SAXS were used. Intensity size distribution curves (DLS) for drug-loaded mPEG113-*b*-P(D,L)LA173 and mPEG113-*b*-P(D,L)LA135 nanoparticles reveal one peak, except for nanoparticles based on mPEG113-*b*-P(D,L)LA62 copolymer with the shortest P(D,L)LA block (Figure S2 of the Supplementary Materials). These two peaks could be attributed to individual nanoparticles with small *Rh* (< 100 nm) and their submicron size aggregates. It should be noted that a fraction of small particles could be undetectable in a mixture with several percent of large particles [47]. In the case of mPEG113-*b*-P(D,L)LA62, the scattering intensity of individual nanoparticles is high enough to estimate their size (Figure S2 of the Supplementary Materials). Therefore, we suppose that the investigated suspension mainly consists of individual oxaliplatin-loaded nanoparticles with a minor fraction of their aggregates.

> The values of *Rh* of oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles are listed in Table 3. The *Rh* values of drug-loaded nanoparticles are almost identical (in the limits of experimental uncertainty) to the values of *Rh* of drug-free nanoparticles (Table 2). Thus, we assume that oxaliplatin loading does not affect the size of the mPEG113-*b*-P(D,L)LA*n* nanoparticles.

> ζ-Potential of oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles was found to be in the range from −16 to −24 mV (Table 3). The values of ζ-potential of drug-loaded nanoparticles are nearly the same as the values for drug-free nanoparticles (Table 2).

> The morphology of mPEG113-*b*-P(D,L)LA*n* nanoparticles remained unchanged with oxaliplatin loading (data not shown).

> Scattering curves from oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles in Log *I*–Log *s* coordinates are presented in Figure 4a. All the SAXS profiles converge to a plateau in the region of *s* < 0.1 nm<sup>−</sup>1, indicating the absence of large aggregates. The profiles also show the secondary maximum in the region 0.3 < *s* < 1 nm<sup>−</sup><sup>1</sup> (Figure 4a), suggesting that the nanoparticles have a spherical well-defined structure with a relatively narrow size distribution. As one can see from Figures 3a and 4a, the drug loading does not affect the shape of SAXS curves. Thus, we sugges<sup>t</sup> that oxaliplatin loading does not affect the structure and size of the mPEG113-*b*-P(D,L)LA*n* nanoparticles.

> Figure 4b shows the SAXS curves for both oxaliplatin-loaded and drug-free mPEG113-*b*-P(D,L)LA*n* nanoparticles in *I*·*s*2–*<sup>s</sup>* coordinates. As one can see from the Kratky plots (Figure 4b), drug loading leads to an increase of the scattering intensity *I(s)* from the mPEG113- *b*-P(D,L)LA*n* nanoparticles. This increment could be attributed to a higher average electron density of nanoparticles with incorporated oxaliplatin compared with drug-free nanoparticles.

> The *Rg* values of oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles determined from the Guinier plots (Figure S3 of the Supplementary Materials) are listed in Table 3. It was found that the *Rg* values of drug-loaded nanoparticles are almost identical (in the limits of experimental uncertainty) to the values of *Rg* of drug-free nanoparticles (Table 2).

The pair distance distribution functions *P(R)* for oxaliplatin-loaded mPEG113-*b*-P(D,L) LA*n* nanoparticles are bell-shaped with a well-defined maximum shifted to distances smaller than *Dmax/2* (Figure S4 of the Supplementary Materials). We assume that the shift of the *P(R)* function maximum can be attributed to "core-corona" structure of oxaliplatinloaded mPEG113-*b*-P(D,L)LA*n* nanoparticles. The values of *R* and *Dmax/2* corresponding to the peak value and the maximum dimension of the scattering objects are listed in Table 3. *Rg* values evaluated from *P(R)* functions were compared with *Dmax/2* and *R* values. The *2Rg/Dmax* values were found to be in the range of 0.61–0.63, whereas *Rg*/*R* was in the range of 0.80–0.95 (Table 3). Thus, one can suppose that oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles also have higher electron density in the inner part than that in the outer part, i.e., a "core-corona" structure [37,48,51].

Based on SAXS data, we estimated the core-corona interface area of mPEG113-*b*-P(D,L)LA*n* nanoparticles *sint* (Table 4). Nanoparticles of mPEG113-*b*-P(D,L)LA62 copolymer with the shortest PLA block have the largest core-corona interface area available for oxaliplatin adsorption (Table 4). In addition, as one can see from Table 4, the value of tethering density of hydrophilic PEG chains *σ* on the surface of PLA core is the highest for the mPEG113-*b*-P(D,L)LA62 particles (detailed description of the calculation can be found in the Supplementary Materials) that is also favorable for oxaliplatin encapsulation. Therefore, mPEG113-*b*-P(D,L)LA62 particles show the highest values of drug loading content and encapsulation efficacy (Table 4). Based on SAXS data, we suppose that oxaliplatin loading does not affect the size and structure of the mPEG113-*b*-P(D,L)LA*n* nanoparticles (Tables 2 and 3). It seems that the amount of loaded drug is insufficient to cause significant changes in these parameters. However, a higher scattering intensity *I(s)* observed for drug-loaded nanoparticles could be attributed to an increase of average electron density of the particles due to oxaliplatin incorporation. Higher initial oxaliplatin loading can lead to a larger drug content in nanoparticles and make it distinguishable on the SAXS curves, which will allow us to investigate the localization of drug in carrier.

**Table 4.** Parameters of oxaliplatin-loaded mPEG113-*b*-P(D,L)LA*n* nanoparticles.


1 The value of PLA core radius evaluated from SAXS (equivalent to the maximum position *R* on the pair distance distribution function *P(R)*). 2 The value of core-corona interface area *sint*. 3 The value of tethering density of PEG chains *σ* on the PLA core surface. 4 The value of oxaliplatin loading content. 5 The value of encapsulation efficacy.

Thus, in the present work, we showed that the highest oxaliplatin loading content at the core-corona interface of mPEG113-*b*-P(D,L)LA*n* nanoparticles through its physical adsorption was 3.8 wt.% with initial drug loading content 5 wt.%. The obtained value is sufficiently higher compared to the value of oxaliplatin loading content in PEG-*b*-PLA nanoparticles reported in ref. [15], i.e., 0.053 wt.%. It should be noted that generally, physical encapsulation of oxaliplatin into polymeric carriers results in lower values of oxaliplatin loading content (3–5 wt.%) [13,26] compared to its content (tens of percent) [14,20–24] after chemical conjugation of oxaliplatin active part with polymer chains. Nevertheless, oxaliplatin-loaded chitosan based polymeric particles showed enhanced cytotoxicity activity against cancer cells compared to free oxaliplatin [26]. Moreover, chitosan-based polymeric micelles and PEG-*b*-PLA nanoparticles with incorporated oxaliplatin are able to selectively accumulate in tumors [15,26].

One of the strategies to enhance the encapsulation efficacy of platinum drugs into polymeric carriers is variation of their lipophilicity. Margiotta et al. investigated the effect of the carboxylate ligand chain length on the encapsulation efficacy of Pt(IV) prodrug complex in poly(lactide-*co*-glycolide)-PEG PLGA-PEG nanoparticles [55]. The authors reported that the amount of Pt atoms encapsulated in PLGA-PEG nanoparticles increased approximately 2 times with an increase of the length of the carboxylate ligand chain from 2 to 10 carbon atoms. In the present work, we studied the effect of mPEG113-*b*-P(D,L)LA*n* copolymers composition on the encapsulation efficacy of oxaliplatin. Based on the obtained results, we sugges<sup>t</sup> that variation of the ratio of hydrophobic and hydrophilic block lengths in mPEG113-*b*-P(D,L)LA*n* copolymers can be another useful strategy to enhance Pt-complex loading content in polymeric nanoparticles without chemical modification of the drug (Table 4).

## **3. Materials and Methods**
