Hydrophobic Modification of Poly(γ-glutamic acid) by Grafting 4-Phenyl-butyl Side Groups for the Encapsulation and Release of Doxorubicin

The delivery of drugs is a great challenge, since most of active pharmaceutical ingredients developed today are hydrophobic and poorly water soluble. From this perspective, drug encapsulation on biodegradable and biocompatible polymers can surpass this problem. Poly(γ-glutamic acid) (PGGA), a bioedible and biocompatible polymer has been chosen for this purpose. Carboxylic side groups of PGGA have been partially esterified with 4-phenyl-butyl bromide, producing a series of aliphatic–aromatic ester derivatives with different hydrophilic–lipophilic balances. Using nanoprecipitation or emulsion/evaporation methods, these copolymers were self-assembled in a water solution, forming nanoparticles with average diameters between 89 and 374 nm and zeta potential values between −13.1 and −49.5 mV. The hydrophobic core containing 4-phenyl-butyl side groups was used for the encapsulation of an anticancer drug, such as Doxorubicin (DOX). The highest encapsulation efficiency was reached for a copolymer derived from PGGA, with a 46 mol% degree of esterification. Drug release studies carried out for 5 days at different pHs (4.2 and 7.4) indicated that DOX was released faster at pH 4.2, revealing the potential of these nanoparticles as chemotherapy agents.


Introduction
Cancer is one of the most lethal diseases these days [1]. The traditional treatment is chemotherapy, in which the anticancer drug is administered through an intravenous injection. The concentration of the drug in the systemic circulation after injection is initially high and subsequently decreases very fast due to hepatic and renal clearances, reducing its therapeutic effect [2]. On the other hand, most anticancer drugs have a low therapeutic window, which causes toxicity in several healthy tissues [3,4]. This problem can be fixed if the drug is administered in a controlled manner through a sustained release on the damaged tissue, using polymers as release regulators [5][6][7].
In the last few decades, scientists have been carrying out many studies for developing new drug delivery systems (DDS) that are able to optimize drug loading and release with a greater long life and effectiveness. Particularly, in the biomedical field, self-assembled systems made of biodegradable amphiphilic polymers at the nanoscale size, such as nanoparticles, polymer micelles, nanotubes, nanogels, and polymersomes, have received a lot of attention [8][9][10]. From this perspective, self-assembled coronal graft or block copolymer nanospheres are appealing systems [11,12]. These systems have the ability to self-assemble in aqueous media, forming nanostructured particles. The preparation and development of these structures are among the issues that biomedical sciences are facing in the field of DDS [13].

Characterization
The 1 H NMR spectra were recorded on a Bruker AMX-300 spectrometer (Billerica, MA, USA) at 25 • C. The 1 H NMR spectra were acquired at 300.1 MHz. Samples were dissolved in deuterated chloroform (CDCl 3 ) or DMSO-d 6 , and the spectra were internally referenced against tetramethylsilane (TMS). Of the sample, 10 mg was dissolved in approximately 1 mL of solvent for 1 H NMR.
Fourier transform infrared spectra (FT-IR) were acquired using a Perkin-Elmer Frontier FT-IR spectrometer (Waltham, MA, USA), provided with a universal-attenuated total reflectance ATR accessory. Infrared spectra were recorded in the 4000-650 cm −1 range at a resolution of 4 cm −1 , and 16 scans were collected. Molecular weights were determined by GPC, using HFIP containing sodium trifluoroacetate (6.8 g·L −1 ) within Waters equipment (Foster City, CA, USA), provided with RI and UV detectors and HR5E and HR2 Waters linear Styragel columns (7.8 mm × 300 mm). Of the sample solution, 0.1 mL (0.1% w/v) was injected and chromatographed with a flow rate of 0.5 mL·min −1 . The molar mass averages and distributions were calibrated against PMMA standards.
Thermogravimetric analyses were carried out under a nitrogen flow rate of 20 mL·min −1 and at a heating rate of 10 • C·min −1 , within a temperature range of 30 to 600 • C, using a Mettler Toledo TGA2 (Columbus, OH, USA). Sample weights of around 5-10 mg were used in these experiments.
Dynamic light scattering studies were performed using a Zetasizer Nano ZS series Malvern instrument (Worcestershire, UK), equipped with a 4 mW He-Ne laser operated at a wavelength of 633 nm. The samples were placed in disposable cuvettes thermostated at 25 • C. The non-invasive back-scatter optical arrangement was used to collect the light scattered by the particles at an angle of 173 • . The particle hydrodynamic sizes and ζ-potential measurements were examined.
Absorbance measurements were examined using a UV-visible spectrophotometer (Cambridge, England, UK) and the samples were dissolved in DMSO. The drug concentration was calculated with a calibration curve obtained from the known amounts of free DOX as standards. SEM images were taken with a field-emission JEOL JSM-7001F (JEOL, Tokyo, Japan) from platinum/palladium-coated samples. The samples were prepared by depositing a drop of the nanoparticle dialysis solution onto a copper surface. Different dilutions were essayed to observe free individual nanoparticles and DOX-encapsulated nanoparticles. The mean diameter of the nanoparticles was determined using the ImageJ software [33].

Esterification of PGGA
PGGA was esterified with 4-phenyl-butyl bromide in solution, using a general procedure reported by Kubota et al. [34]. Specifically, 500 mg (4.0 mmol) PGGA was dissolved in 100 mL of NMP and left under stirring at 80 • C for 1 h, for the complete dissolution of the polymer. Afterwards the solution was cooled down to 60 • C and variable amounts of NaHCO 3 were added to the solution, depending on the degree of esterification. After that, 4-phenyl-butyl bromide was slowly added at a necessary amount, to reach the desired conversion. The reaction was left to proceed for 48 h, until no evolution was observed in the reaction and the esterified polymer was recovered by precipitation in acidic water. Then, the copolymer was washed with neutral water and dried under vacuum for 24 h. The copolymers obtained were named PGGAH x PhB y , x and y being the molar ratio (%) of unmodified and modified repeating units.

Nanoparticle Preparation
Two different methods were assayed to prepare the nanoparticles: (1) dialysis/precipitation (nanoprecipitation) and (2) emulsion/evaporation (nanoemulsion). In the first method, 5 mg of the copolymer was dissolved in 1 mL of NMP, and afterward, 1 mL of distilled water was added dropwise under magnetic stirring. The solution was introduced in a dialysis bag of cellulose, with a molecular weight cut-off of 6000-8000 Da, and was dialyzed for 24 h at room temperature. Distilled water was replaced four times at 2, 5, 9, and 17 h, to remove any residual NMP solvent. The second emulsion/evaporation method was also assayed for copolymers with higher degrees of esterification. Briefly, 5 mg of copolymer was dissolved in 1 mL DCM, and this solution was added to 10 mL of 0.5% poly(vinyl alcohol) (PVA) aqueous solution. The mixture was emulsified with the help of an ultrasounds bath for 45 s (three times). Then, this solution was dispersed in 20 mL of water under magnetic stirring and DCM was rotary-evaporated.
Particle average diameters, distributions and surface charges of nanoparticles were determined by DLS.

Stability of Nanoparticles in Solution
After producing nanoparticles, they were kept in solution at 2-4 • C for 4 weeks. The effect of storage time on the stability of the dispersion was evaluated.

Doxorubicin Loading and Releasing
Doxorubicin hydrochloride (DOX·HCl) was used as a drug model in this study. Of the DOX·HCl, 2 mg was dissolved in 2 mL of DMSO, and then 20 µL of TEA was added, leaving the solution for 24 h under magnetic stirring in a dark room at room temperature. TEA was added in order to remove the HCl from the DOX salt, enhancing drug encapsulation [35]. On the other hand, the PGGAH x PhB y copolymer (10 mg) was solubilized in 1 mL of DMSO. Afterward, the two solutions were mixed and 1 mL of deionized water was added dropwise and left under magnetic stirring for 2 h. This solution was then dialyzed against 1 L of distilled water to remove the free DOX, using a cellulose membrane MWCO 6000-8000 kDa. After 24 h, half of the dialysis bag was lyophilized. The weighted amount of loaded nanoparticles was dissolved in DMSO, and the content of the drug was determined via UV-Vis spectroscopy using a correct blank and a calibration curve.
The drug loading (DL) and encapsulation efficiency (EE) were determined using the following formulas: % DL = (mass of the DOX loaded into NP/total mass of DOX-loaded NP) × 100 (1) % EE = (mass of the DOX loaded into NP/mass of DOX added initially) × 100 (2) Regarding in vitro release studies, the DOX-loaded nanoparticles were incubated in two aqueous buffers (PBS pH 7.4, citrate-phosphate pH 4.2) under simulated physiological conditions, and half of the solution that had not been lyophilized was placed in a dialysis bag (MWCO 6000-8000 kDa), which was then immersed in 20 mL of buffer and kept under magnetic stirring at 37 • C. For measuring the amount of the drug released, 1.5 mL aliquots were taken out from the releasing medium at scheduled times, and the solution was replaced with an equal volume of a fresh medium. The amount of the released DOX was carried out by absorption spectrometry at λ max (480 nm) using a UV-Vis spectrophotometer [36].

PGGA Esterification
Esterification of PGGA with 4-phenyl-butyl bromide was carried out according to the method previously reported by Kubota et al. [34] (Figure 1). By varying the concentrations of PGGA, 4-phenyl-butyl bromide, and NaHCO 3 , PGGAH x PhB y copolymers with different degrees of esterification were obtained.  Table 1 displays the ratio of reactants used in the feed and the degree of esterific determined via 1 H NMR, the yields and average molar masses for all PGGAHxPhB polymers prepared. The copolymers were recovered at high yields (55-97% white-to-yellow powders.   copolymers prepared. The copolymers were recovered at high yields (55-97%), as white-toyellow powders. 1 PGGA: 4-phenyl-butyl bromide-NaHCO 3 ratios. 2 Degree of esterification of PGGA calculated via 1 H-NMR; 3 Weight-average molecular weight and dispersity determined via GPC (PGGAH x PhB y copolymers with a low degree of esterification were insoluble in HFIP).

Characterization of Copolymers
The weight-average molecular weight of PGGAH x PhB y increased almost continuously with the degree of esterification, and all the copolymers assayed showed dispersities between 1.5 and 1.9 (Table 1). 1 H NMR was used to monitor the reaction and determine the degree of esterification achieved (Figure 2a). PGGA displayed four signals from a down-to an up-field shift corresponding to the NH (a, 7.6 ppm), CH (b, 4.2 ppm), α-CH 2 (c, 2.2 ppm), and β-CH 2 (d, 1.9 ppm); the last one split due to the presence of an asymmetric center in the repeating unit. On the other hand, copolymers obtained via the esterification of carboxylic groups displayed new peaks at 8.3 ppm (NH, a ), 7.2 (Ar-H, i), 4.0 ppm (OCH 2 , e), 2.6 ppm (CH 2 , h), and 1.6 ppm (CH 2 , f and g). Through the integration of signals due to the α-CH 2 and aromatic protons, the degree of esterification was calculated. Figure 2b shows the FTIR spectra of PGGA and two PGGAH x PhB y copolymers with an increasing content of the phenyl-butyl side groups. The spectrum of PGGA shows a band centered at 3288 cm −1 corresponding to the NH stretching vibration of the amide group. The peak at 1720 cm −1 is associated with the stretching vibration of the carbonyl of the COOH side groups, and a small shoulder at 1640 cm −1 is due to the stretching vibration of the CO amide group (amide I). When PGGA is partially esterified, it can be observed that the signal of the carbonyl group shifts to higher frequencies, appearing at 1732 and 1736 cm −1 for PGGAH 37 PhB 63 and PGGAH 3 PhB 97 , respectively. This displacement is mainly caused by a reduction in intermolecular hydrogen bonding interactions. Additionally, the appearance of a new peak at 1174-1180 cm −1 can be clearly observed on partially esterified PGGA, and was correlated with the C-O stretching vibration. On the other hand, the presence of aromatic groups can be easily identified by the absorption bands at 3027 cm −1 corresponding to the Ar-H stretching vibrations, and at 746 and 698 cm −1 corresponding to out-of-plane Ar-H bending vibrations. It can be concluded that FTIR spectroscopy is a complementary technique to 1 H NMR, that allows for the determination of the degree of esterification of the copolymers obtained, at least qualitatively.
other hand, the presence of aromatic groups can be easily identified by the absorption bands at 3027 cm −1 corresponding to the Ar-H stretching vibrations, and at 746 and 698 cm −1 corresponding to out-of-plane Ar-H bending vibrations. It can be concluded that FTIR spectroscopy is a complementary technique to 1 H NMR, that allows for the determination of the degree of esterification of the copolymers obtained, at least qualitatively. The thermal stability of PGGA and PGGAHxPhBy copolymers was evaluated via TGA, and data collected from these thermograms are collated in Table 2. The weight loss, concomitant to degradation, occurs between 200 °C and 330 °C. The residual material left at the end of the test decreases in most PGGAHxPhBy derivatives with a higher degree of modification.
As can be observed, the decomposition process for most copolymers involves multi-step weight losses, with the main decomposition step taking place between 294 and 332 °C. As an example, the thermal behaviors of PGGA, PGGAH54PHB46, and PGGAH3PhB97 are displayed in Figure 3.  The thermal stability of PGGA and PGGAH x PhB y copolymers was evaluated via TGA, and data collected from these thermograms are collated in Table 2. The weight loss, concomitant to degradation, occurs between 200 • C and 330 • C. The residual material left at the end of the test decreases in most PGGAH x PhB y derivatives with a higher degree of modification. 1 Onset decomposition temperature measured at 10% of loss of the initial we decomposition temperature. In bold the temperature of main decompositio weight at 600 °C.

Preparation, Characterization, Morphology, and Stability of PGGAHxPh over Time
Partial and almost-full modification of PGGA via the esterification side groups resulted in amphiphilic copolymers that were able to fo nanostructures. As can be observed in Table 3, all copolymers were ab of nanometric sizes using both nanoprecipitation and nanoemulsion m ticles with average diameters below 200 nm and good polydispersities copolymers with intermediate compositions or copolymers with a hig fication. Comparing both methods, it was observed that nanoparticl could be obtained by nanoprecipitation. On the other hand, all nanopa negative ζ-potential attributed to the remaining carboxylic side group at the surface of the nanoparticles, and were ionized at the neutral pH for the dialysis.
Assays to determine their morphology were performed via SEM ure 4, all particles displayed spherical shapes and nanometric sizes, h drodynamic diameters between 156 and 234 nm ( Figure S1).

Preparation, Characterization, Morphology, and Stability of PGGAH x PhB y Nanoparticles over Time
Partial and almost-full modification of PGGA via the esterification of the carboxylate side groups resulted in amphiphilic copolymers that were able to form self-assembled nanostructures. As can be observed in Table 3, all copolymers were able to form particles of nanometric sizes using both nanoprecipitation and nanoemulsion methods. Nanoparticles with average diameters below 200 nm and good polydispersities were obtained for copolymers with intermediate compositions or copolymers with a high degree of esterification. Comparing both methods, it was observed that nanoparticles of smaller sizes could be obtained by nanoprecipitation. On the other hand, all nanoparticles displayed a negative ζ-potential attributed to the remaining carboxylic side groups that were placed at the surface of the nanoparticles, and were ionized at the neutral pH of the water used for the dialysis. Assays to determine their morphology were performed via SEM. As shown in Figure 4, all particles displayed spherical shapes and nanometric sizes, having average hydrodynamic diameters between 156 and 234 nm ( Figure S1).  The stability of PGGAHxPhBy nanoparticles in solution was assessed, maintaining the dispersions over a 4-week period at low temperatures (2-4 °C). As representative examples, three copolymer compositions were assayed, and their stability remained almost unaltered. No precipitation was observed in any sample and the average diameter, as well as polydispersities, remained very stable for this period of time (Table 4 and Figure 5). This good stability can be caused by the small sizes and the high surface charges that the nanoparticles present, which prevents their agglomeration. As can be observed, all nanoparticles displayed high negative ζ-potential values over the period of storage. Only PGGAH54PhB46 nanoparticles displayed a small reduction in their average diameter after 4 weeks of storage. This striking behavior could be caused by a compaction of the nanoparticles, favored by the hydrophobic interactions and the temperature used during storage. The stability of PGGAH x PhB y nanoparticles in solution was assessed, maintaining the dispersions over a 4-week period at low temperatures (2-4 • C). As representative examples, three copolymer compositions were assayed, and their stability remained almost unaltered. No precipitation was observed in any sample and the average diameter, as well as polydispersities, remained very stable for this period of time (Table 4 and Figure 5). This good stability can be caused by the small sizes and the high surface charges that the nanoparticles present, which prevents their agglomeration. As can be observed, all nanoparticles displayed high negative ζ-potential values over the period of storage. Only PGGAH 54 PhB 46 nanoparticles displayed a small reduction in their average diameter after 4 weeks of storage. This striking behavior could be caused by a compaction of the nanoparticles, favored by the hydrophobic interactions and the temperature used during storage.

Doxorubicin Loading and Encapsulation Efficiency
Doxorubicin (DOX) is an outstanding amphiphilic drug that is commonly used in cancer treatment [37]. In order to encapsulate DOX in nanoparticles, the DOX·ClH was previously converted into DOX, and then added to the initial copolymer solution. Nanoparticles were produced by the nanoprecipitation method, and the hydrophobic drug was then encapsulated [38]. In order to check the effect of the copolymer composition on the drug loading and encapsulation efficiency, nanoparticles were prepared from four different copolymers, covering all degrees of esterification (Table 5).

Doxorubicin Loading and Encapsulation Efficiency
Doxorubicin (DOX) is an outstanding amphiphilic drug that is commonly used in cancer treatment [37]. In order to encapsulate DOX in nanoparticles, the DOX·ClH was previously converted into DOX, and then added to the initial copolymer solution. Nanoparticles were produced by the nanoprecipitation method, and the hydrophobic drug was then encapsulated [38]. In order to check the effect of the copolymer composition on the drug loading and encapsulation efficiency, nanoparticles were prepared from four different copolymers, covering all degrees of esterification (Table 5). small changes in the pH or ionic strength [39][40][41][42]. In this work, DOX was transformed in its neutral form through the addition of TEA, allowing for the entering into the core of the nanoparticle. As shown in Table 5, after loading the drug, it was observed that the ζ-potential increased, indicating that there was no neutralization of the surface charge of the nanoparticle; the drug is actually enclosed in the hydrophobic core of the nanoparticle created by the phenyl-butyl side groups grafted in the PGGA. As can be observed, the PGGAH 54 PhB 46 copolymer displayed higher ζ-potential values than PGGAH 70 PhB 30 in both loaded and unloaded nanoparticles. Although the number of carboxylate groups is lower in the former copolymer, it seems that it could self-assemble better, exposing greater amounts of carboxylate groups outside the nanoparticles. On the other hand, after loading with DOX, the nanoparticles obtained from PGGAH 89 PhB 11 displayed micrometric average diameters. It seems that the low content of hydrophobic groups in this copolymer requires a higher number of polymeric chains to stabilize the drug inside the particle. The amount of drug used in all four samples was the same and equal to 2 ± 0.2 mg, but according to DL-and EE-obtained values, (Table 5), the maximum EE of DOX under neutral conditions after one day of incubation was 46%, obtained for the PGGAH 54 PhB 46 copolymer. SEM images were collected in order to determine the morphology of the nanoparticles obtained. Quasy-spherical structures corresponding to the nanoparticles loaded with DOX, with average hydrodynamic diameters between 165 and 175 nm, were observed, verifying that the morphology is maintained in relation to the nanoparticles not loaded with DOX ( Figure 6 and Figure S2).
When DOX is used as the hydrochloric acid salt (DOX·HCl), the electrostatic interactions between the drug and the polymer with carboxylic groups maintain the drug at the surface of the nanoparticle. However, these interactions can be easily broken by small changes in the pH or ionic strength [39][40][41][42]. In this work, DOX was transformed in its neutral form through the addition of TEA, allowing for the entering into the core of the nanoparticle. As shown in Table 5, after loading the drug, it was observed that the ζ-potential increased, indicating that there was no neutralization of the surface charge of the nanoparticle; the drug is actually enclosed in the hydrophobic core of the nanoparticle created by the phenyl-butyl side groups grafted in the PGGA. As can be observed, the PGGAH54PhB46 copolymer displayed higher ζ-potential values than PGGAH70PhB30 in both loaded and unloaded nanoparticles. Although the number of carboxylate groups is lower in the former copolymer, it seems that it could self-assemble better, exposing greater amounts of carboxylate groups outside the nanoparticles. On the other hand, after loading with DOX, the nanoparticles obtained from PGGAH89PhB11 displayed micrometric average diameters. It seems that the low content of hydrophobic groups in this copolymer requires a higher number of polymeric chains to stabilize the drug inside the particle.
The amount of drug used in all four samples was the same and equal to 2 ± 0.2 mg, but according to DL-and EE-obtained values, (Table 5), the maximum EE of DOX under neutral conditions after one day of incubation was 46%, obtained for the PGGAH54PhB46 copolymer. SEM images were collected in order to determine the morphology of the nanoparticles obtained. Quasy-spherical structures corresponding to the nanoparticles loaded with DOX, with average hydrodynamic diameters between 165 and 175 nm, were observed, verifying that the morphology is maintained in relation to the nanoparticles not loaded with DOX ( Figures 6 and S2).

In Vitro Drug Release Behavior of NPs
Considering that the fast release in the body minimizes the effects of drugs and has an adverse effect on organs [43][44][45], one of the purposes of this research is to study the effect of PGGA esterification on the drug release behavior of nanoparticles. PBS, with a

In Vitro Drug Release Behavior of NPs
Considering that the fast release in the body minimizes the effects of drugs and has an adverse effect on organs [43][44][45], one of the purposes of this research is to study the effect of PGGA esterification on the drug release behavior of nanoparticles. PBS, with a pH of 7.4 (mimicking the pH of a normal human blood) and a citrate-phosphate pH of 4.2 (lysosomal pH), were used to study the release of DOX in NP. The release curves obtained from the nanoparticles prepared with the two representative PGGAH x PhB y copolymers are shown in Figure 7.
Nanoparticles sensitive to pH, such as those obtained here, can release drugs quickly at the tumor site and very slowly in the peripheral circulation [49][50][51][52][53][54]. The optimization of nanoparticles through PEGylation and adding targeting ligands is a work that is planned to be carried out by us in the near future.

Conclusions
Amphiphilic copolymers have been obtained via the partial esterification of bacterial poly(γ-glutamic acid) with 4-phenyl-butyl bromide. These copolymers were able to Nanoparticles prepared from the PGGAH 70 PhB 30 and PGGAH 54 PhB 46 copolymers release 38% and 21% DOX, respectively, in the first 5 h at a pH of 7.4. As expected, the release is more sustained for nanoparticles obtained from the copolymer with a higher content of phenyl-butyl side groups, since it will have a greater ability to interact with the DOX hydrophobic drug. Surprisingly, the trend was reversed in the release profile at a pH of 4.2, and the release rate was greater for the copolymer with the higher degree of esterification. The in vitro maximum release of DOX was almost 100% for PGGAH 54 PhB 46 and 95% for PGGAH 70 PhB 30 . A combined effect of a greater destabilization of the nanoparticle, due to the partial loss of the surface charge and the protonation of the amino groups of the DOX, may be the cause of this behavior. Contrastingly, the higher release rate noticed in acid media has also been observed in the micelles of other hydrophobically modified polypeptides, such as poly(α,β-aspartic acid) and PEG-grafted poly(α-glutamic acid), and attributed to the formation of agglomerates in the latter case, that release the cargo and are caused by a reduction in the repulsion charges [46,47]. In our case, we believe that water can swell the nanoparticle and, in the case of having an acid release medium, the amino group of the DOX can become protonated, thus increasing its solubility, allowing its faster diffusion from the nanoparticle to the medium.
Drug release profiles have been fitted to different kinetic models (zero-order, firstorder, Higuchi, and Korsmeyer-Peppas models, Table S1) [48]. It was observed that the best fit of the release profile was achieved with the Korsmeyer-Peppas model. Since n < 0.45 for nanoparticles obtained from PGGAH 70 PhB 30 at both pH conditions, a Fickian diffusion mechanism of drug, outward the nanoparticles, was suggested. On the other hand, values of 0.74 and 0.65 n were obtained at pHs 7.4 and 4.2, respectively, for the DOX-loaded nanoparticles made of PGGAH 54 PhB 46 , indicating an anomalous (non-Fickian) diffusion behavior for this copolymer composition.
Nanoparticles sensitive to pH, such as those obtained here, can release drugs quickly at the tumor site and very slowly in the peripheral circulation [49][50][51][52][53][54]. The optimization of nanoparticles through PEGylation and adding targeting ligands is a work that is planned to be carried out by us in the near future.

Conclusions
Amphiphilic copolymers have been obtained via the partial esterification of bacterial poly(γ-glutamic acid) with 4-phenyl-butyl bromide. These copolymers were able to selfassemble into spherical nanoparticles with average diameters of around 200 nm, using nanoprecipitation and nanoemulsion methods. The hydrophobic core was composed of repeating units containing the phenyl-butyl ester groups and the hydrophilic shell, composed of repeating units with unreacted carboxylate groups. These nanoparticles were able to encapsulate Doxorubicin at a high encapsulation efficiency, and release it faster at acidic pHs. These results indicate that the modified PGGA copolymers can be used for preparing nanoparticles that act as anti-cancer drug carriers of hydrophobic drugs, such as DOX.