Redox-Responsive Disulfide Cross-Linked PLA PEG Nanoparticles

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1 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. pubs.acs.org/macromolecules Redox-Responsive Disulfide Cross-Linked PLA PEG Nanoparticles Tiziana Fuoco, Daniela Pappalardo,*,, and Anna Finne-Wistrand*, Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, Stockholm, Sweden Department of Science and Technology, University of Sannio, via dei Mulini 59/A, Benevento, Italy *S Supporting Information Downloaded via on November 20, 2018 at 20:56:17 (UTC). See for options on how to legitimately share published articles. ABSTRACT: We have developed a strategy for the preparation of redox-responsive PEG PLA-based nanoparticles containing disulfide bonds that can be disassembled in the presence of cellular concentrations of glutathione. Functionalized poly- (lactide)s were prepared by ring-opening copolymerization of L- lactide and 3-methyl-6-(tritylthiomethyl)-1,4-dioxane-2,5-dione, a monomer bearing a pendant trityl-thiol group, followed by the postpolymerization modification of trityl-thiol into pyridyl disulfide groups. Polymeric networks composed of PLA and PEG blocks linked by disulfide bonds were prepared by a disulfide exchange reaction between the functionalized PLAs and telechelic PEG having thiol groups at both ends, HS-PEG-SH, in DMF. When dialyzed against water, they assembled into dispersible nanoparticles, with a flowerlike structure having a hydrophobic core and a hydrophilic shell, with sizes in the range nm that are suitable for drug delivery. The effects of the number of functional groups, molecular weight, and concentration on the nanoparticle size were evaluated. The stability of the nanoparticles after dilution and the redox-responsive behavior in the presence of different concentrations of glutathione were assessed. The hydrophobic molecule Nile red could be encapsulated in the nanoparticles and then released in the presence of glutathione at cellular concentration. INTRODUCTION or dispersible copolymers Block copolymers of PEG and Copolymers having both poly(ethylene glycol) (PEG) and aliphatic polyesters are amphiphilic and can assemble into aliphatic polyester components have become appealing base micellar structures with a hydrophobic compact inner core and polymers for biomedical applications. PEG is an important type a hydrophilic swollen outer shell in aqueous environments. of biocompatible and water-soluble polymer, and pegylation Owing to their core shell structure, micelles can easily can increase the blood circulation time of drug carriers. incorporate hydrophobic drugs, and the resulting increased However, because of its nondegradability, the use of low-molarmass PEGs is preferable. It has been found that PEG samples drug concentration in the body fluid can achieve a higher efficacy. 11 However, the instability problems caused by dilution with molecular weights of 1 and 6 kda were excreted within 12 effects when micelles are injected in the body are the major h in humans. Because of the absence of functional groups along drawback for micelles as drug carriers. the chains, only terminal functionalities are available for further Several approaches have been developed to contrast these modification or conjugation to biological motifs. 1 instabilities. We have previously evaluated tree-shaped On the other hand, aliphatic polyesters, typically used in copolymers based on PEG and poly(lactide) (PLA) for micelle biocompatible devices, are hydrophobic polymers which can formation and demonstrated that the physical properties of degrade through an enzymatic route or by hydrolytically micelles, such as the critical micellar concentration (CMC), size cleavage of the ester bonds. Contrary to PEG, multiple distribution, and their functions, can be tailored by diversifying functionalities can be introduced to aliphatic polyesters either the number, structure, and tacticity of the PLA arms. 12 via polymerization using functionalized ester-based monomers Furthermore, the micelle size and stability can be modulated or via postpolymerization modification. 2 5 For this purpose, we by using linear block copolymers made of PEG and aliphatic recently designed and synthesized a lactide-type monomer polyesters of various compositions by controlling the bearing a latent thiol group, which is a versatile building block crystallinity of the core. 13 Other approaches to stabilize for the preparation of functionalized aliphatic polyesters by micelles, such as cross-linking of the polymeric chains, have ring-opening polymerization. 6 Notably, thiol chemistry is an been presented. Harth et al. described the synthesis of welldefined degradable nanoparticles in terms of their size efficient tool for polymer synthesis and postpolymerization reactions, tailoring the properties and applications of polymeric distribution and dimensions, prepared through a controlled materials. 7 Combinations of PEG and aliphatic polyester merge the Received: June 21, 2017 above-mentioned properties, and they have been largely Revised: August 14, 2017 exploited to provide a variety of multivalent and water-soluble Published: September 7, American Chemical Society 7052

2 Table 1. Synthesis of Poly[(TrtS-LA)-co-LA] and Postpolymerization Modification to Poly[(PDS-LA)-co-LA] a feed (mol %) conversion (%) composition (mol %) poly[(pds-la)-co-la] poly[(pds-la)-co-la] sample TrtS-LA LA t (h) TrtS-LA LA TrtS-LA LA yield (%) M n (kda) c Đ c M n (kda) c Đ c P P2a P2b P a Polymerization conditions: [monomers]:[sn(oct) 2 ]:[ethylene glycol] = 1000:1:4; M n,th 80 kda; T = 110 C. b Determined by 1 H NMR spectra (CDCl 3, RT). c Determined by SEC in CHCl 3, versus narrow polystyrene standards. interchain/intrachain cross-linking process between functionalized aliphatic polyester and a cross-linking agent. 14 We reasoned that the use of dynamic covalent cross-links could result in the formation of structurally adaptive nanoparticles that are able to respond to external stimuli. 15 Notably, materials incorporating disulfide linkages have been shown to hold great promise for tumor-targeted drug delivery applications. Indeed, disulfide bonds are stable under the normal body temperature, ph, and oxidation environment and can be reduced to thiols by reducing agents such as glutathione (GSH). Interestingly, there is a redox potential difference between the inside and outside of cells. The concentration of intracellular GSH ( mmol L 1 ) is approximately 200 times higher than that of the extracellular GSH (1 20 μmol L 1 ). 16 The extracellular GSH is insufficient for reducing disulfide bonds. Accordingly, once the polymeric carriers containing disulfide bonds enter the target cells by endocytosis, they release drugs quickly and effectively owing to the reductive cleavage of the disulfide bonds reduced by GSH Here, a new strategy to create PLA PEG-based nanoparticles containing disulfide bonds has been developed using a disulfide exchange reaction between PLA chains bearing pyridyl disulfide groups and telechelic PEG chains bearing thiol groups at both ends, SH-PEG-SH. The effects of the molecular weight, number of functional groups, concentration, and solvent on the size of the nanoparticles were investigated. Moreover, the nanoparticle stability after dilution and the redox response were evaluated in the presence of GSH. In vitro tests of drug loading and release of the Nile red as a hydrophobic model of guest molecule were also carried out and are discussed herein. EXPERIMENTAL SECTION Materials and Methods. The moisture- and air-sensitive materials were manipulated under nitrogen using Schlenk techniques or using an MBraun 150B glovebox. Before use, the glassware was dried overnight in an oven at 120 C. Dichloromethane (CH 2 Cl 2 ) was refluxed over CaH 2 and distilled under nitrogen. Stannous octanoate (SnOct 2 ) was purchased from Sigma-Aldrich, dried over molecular sieves, and stored in a glovebox before use. Ethylene glycol, L-lactide (LA), and poly(ethylene glycol) dithiol (HS-PEG-SH; M n = 1000 Da) were purchased from Sigma-Aldrich and used as received. The 3-methyl-6-(tritylthiomethyl)-1,4-dioxane- 2,5-dione (TrtS-LA) was prepared as reported. 6 All other reagents and solvents were purchased from Sigma-Aldrich and used as received. The solvents and chemicals were used as received unless stated otherwise. Dialysis membranes with a molecular weight cutoff between 100 and 500 Da and a volume per length ratio of 3.1 ml cm 1 were obtained from Spectrum Laboratories. Instruments and Measurements. NMR spectra were obtained in CDCl 3 at room temperature using a Bruker Avance 400 spectrometer ( 1 H: MHz; 13 C: MHz). The resonances and coupling constants are reported in ppm (δ) and Hz (J), and they are referenced to the residual solvent peak versus Si(CH 3 ) 4 at δ 7.26 ( 1 H) and δ ( 13 C). Spectra were recorded using Bruker TopSpin v2.1 software. Data processing was performed using MestReNova v9.0.0 software. Molecular weights (M n and M w ) and molecular weight dispersities (Đ) were measured by size exclusion chromatography (SEC). The measurements were performed at 30 C on a Verotech PL-GPC 50 Plus system equipped with two PLgel 5 μm MIXED-D ( mm) columns, a PL-RI detector (Varian, Germany), and a PL-GPC 50 Plus autosampler using CHCl 3 as the eluent (1.0 ml min 1 ). Narrow polystyrene standards were used as a reference, and the flow rate fluctuations were corrected using toluene as an internal standard. The glass transition temperatures (T g ), melting points (T m ), and enthalpies of fusion (ΔH m ) of the polymer samples were measured by differential scanning calorimetry (DSC) using aluminum pans and a DSC 2920 TA Instruments apparatus, calibrated with indium. Measurements were performed under nitrogen flow with a heating rate of 10 C min 1 from 80 to +200 C. DSC data were processed with TA Universal Analysis v2.3 software and are reported for the second heating cycle. UV spectra were recorded on a UV vis spectrophotometer 2550 over a range of nm, and the UV data were processed with UV Probe software. Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer Nano ZS at 25 C and at a scattering angle of 90. The measurements were performed both in DMF and in water; the viscosity (η) and the refractive index of the dispersant at 25 C were settled accordingly: DMF (η = mpa s; RI = 1.428); water (η = mpa s; RI = 1.330). The intensity-weighted mean values of the diameter and polydispersity index (PDI) were reported as the average of three measurements. For each measurement, 12 scans were recorded, and the reported error represents the average error incurred during the three measurements. Scanning electron microscopy (SEM) was performed on an FE SEM Hitachi S-4800 with an accelerating voltage of 10.0 kv. Samples were sputter-coated with a layer of platinum. Fluorescence measurements were performed on a Tecan Infinite 200 PRO multifunctional microplate reader with an excitation wavelength = 535 nm. Emission intensity spectra were recorded in the range nm with a step of 5 nm. Copolymerization of 3-Methyl-6-(tritylthiomethyl)-1,4-dioxane-2,5-dione (TrtS-LA) with L-Lactide (LA) To Produce Poly[(TrtS-LA)-co-LA]. A typical polymerization is described herein for sample P1 in Table 1. A previously silanized 25 ml round-bottom flask was charged with SnOct 2 (5.0 mg, 12 μmol), ethylene glycol (3.0 mg, 50 μmol), LA (1.650 g, 11.4 mmol), and TrtS-LA (0.250 g, 0.60 mmol). The polymerization mixture was thermostated at 110 C and magnetically stirred for 24 h. Then, the mixture was cooled to room temperature. The crude copolymer was dissolved in CH 2 Cl 2 and precipitated twice in cold MeOH. The precipitate was filtered, washed sequentially with MeOH, and dried in vacuo at 30 C overnight. The yield was 76 %. Poly[(TrtS-LA)-co-LA]: 1 H NMR (400 MHz; CDCl 3 ) δ (m, 6H, ArH), (m, 9H, ArH), 5.16 (q, 3 J = 7.2 Hz, 2H, CHCH 3 ), 5.01 (q, 3 J = 7.0 Hz, 1H, CHCH 3 O C(O)- CHCH 2 S ), 4.51 (dd, 3 J = 9.2 Hz, 3 J = 3.2 Hz, 1H, CHCH 2 S ), (m, 1H, CHCH 2 S ), (m, 1H, CHCH 2 S ), 1.58 (d, 3 J = 7.2 Hz, 3H, CHCH 3 ), and 1.47 (d, 3 J = 7.0 Hz, 3H, 7053

3 Table 2. DLS Data of the Nanoparticles Obtained for Copolymers Having Different Numbers of PDS-LA Functional Units DMF H 2 O poly[(pds-la)-co-la] PDS-LA units (mol %) a M n, SEC of poly[(pds-la)-co-la] (kda) b diam (nm) c PDI c diam (nm) c PDI c P ± ± ± ± 0.02 P2a ± ± ± ± 0.02 P2b ± ± ± ± 0.02 P ± ± ± ± 0.12 a Determined by 1 H NMR spectra (CDCl 3, rt). b Determined by SEC in CHCl 3, versus narrow polystyrene standards. c Determined by DLS (25 C). Table 3. DLS Data of the Nanoparticles Obtained with Copolymers P1 and P2a at Different Concentrations in DMF and after Dialysis in H 2 O DMF H 2 O poly[(pds-la)-co-la] run C (mg ml 1 ) a diameter (nm) b PDI b diameter (nm) b PDI b P ± ± ± ± 0.02 P ± ± ± ± 0.01 P ± ± ± ± 0.01 P ± ± ± ± 0.01 P ± ± 0.06 n.d. c n.d. c P2a ± ± ± ± 0.05 P2a ± ± ± ± 0.01 P2a ± ± ± ± 0.01 P2a ± ± ± ± 0.02 P2a ± ± ± ± 0.01 P2a ± ± ± ± 0.01 a C is the total polymer concentration in DMF. b Determined by DLS (25 C). c n.d. = not determined. CHCH 3 ). 13 C NMR (100 MHz; CDCl 3 ) δ 169.7, 169.4, 169.3, and ( OC(O)CHCH 3 ), ( OC(O)CHCH 2 S ), 144.3, 129.7, 128.2, and ( C Ar ), 72.0 ( CHCH 2 S ), 69.1 ( CHCH 3 ), 67.6 ( SC(Ph) 3 ), 32.7 ( CHCH 2 S ), and 16.8 ( CHCH 3 ). Modification of Poly[(TrtS-LA)-co-LA] in Poly[(PDS LA)-co- LA]. The reaction was performed as previously reported; 6 a typical procedure is described here for sample P1 (Table 1). The poly(trts- LA)-co-LA (1.03 g, 0.33 mmol of TrtS- groups), prepared as described above, was reacted overnight with trifluoroacetic acid (TFA; 0.20 ml, 2.30 mmol) and Et 3 SiH (0.20 ml, 0.10 mmol) in 7 ml of CH 2 Cl 2. Then, the reaction mixture was dried in vacuo, and the crude polymer was dissolved in 30.0 ml of CH 2 Cl 2. The solution of the crude polymer was added dropwise over 1 h to a solution of 2,2 -dipyridyl disulfide (0.365 g, 1.65 mmol) in 5 ml of CH 2 Cl 2. The reaction mixture was stirred for 3 h, and the poly[(pds-la)-co-la] was collected by precipitation in n-heptane/meoh (95:5) with a yield of 85% (Scheme 2). Poly[(PDS-LA)-co-LA]: 1 H NMR (400 MHz; CDCl 3 ) δ 8.47 (d, J = 4.5 Hz, 1H, pyridyl H), (m, 2H, pyridyl H), (m, 1H, pyridyl H), 5.40 (overlapped signals: m, 2H, CHCH 3 O C(O)CHCH 2 S, CHCH 2 S ), (m, 2H, CHCH 3 O ), (m, 2H, CHCH 2 S ), and 1.60 (d, J = 7.1 Hz, 3H, CHCH 3 ). 13 C NMR (100 MHz; CDCl 3 ) δ 169.7, ( OC(O)- CHCH 3 ), ( OC(O)CHCH 2 S ), 159.3, 149.8, 137.6, 121.4, and ( C Ar ), 71.5, 69.7, and 69.2 ( CH ), 40.4 ( CHCH 2 S ), and 16.8 ( CHCH 3 ). Nanoparticles Preparation. A typical procedure is described herein for the sample prepared with copolymer P2a at a concentration of 10.0 mg ml 1 (Table 2). Poly[(PDS-LA)-co-LA) (P2a, 20 mg; 24 μmol of PDS groups) was dissolved in 2.0 ml of dimethylformamide (DMF). Then, a solution of HS-PEG-SH (12 mg, 24 μmol of SH groups) in 1.2 ml of DMF was added. The reaction was stirred at room temperature for 24 h and followed by UV spectroscopy. An aliquot of 0.1 ml was taken and diluted to 1.0 ml, and the UV spectra were immediately recorded. The concentration of the released 2-thiolpyridone was then calculated knowing that it has a maximum of absorbance at 375 nm in DMF and the value of ε = mol 1 cm 1. The nanoparticles solutions in DMF were then dialyzed against deionized water. Dialysis membranes with a molecular weight cutoff between 100 and 500 Da and a volume per length ratio of 3.1 ml cm 1 were obtained from Spectrum Laboratories. Prior to use, the membrane was soaked in deionized (DI) H 2 O for 15 min. Dialysis of the nanoparticles was achieved by pouring the DMF solution into the dialysis membrane. The membrane was subsequently submerged and dialyzed against 2 L of DI H 2 O under mild stirring for 24 h. During the first 2 h, the dialysis solution was replaced with fresh DI H 2 O every 30 min. Subsequently, for the next 4 h, the water was replaced every hour. Then, dialysis was allowed to occur overnight. The nanoparticles size was determined by DLS both in the DMF solution and in the water phase. In Situ NMR Study of the Nanoparticles Preparation. A NMR tube was filled with HS-PEG-SH (3.0 mg, 6 μmol of SH groups) and 0.8 ml of CDCl 3. Then, the 1 H NMR spectra were recorded. Afterward, poly[(pds-la)-co-la) (P2a, 5.0 mg, 6 μmol of PDS groups) was added to the NMR tube, and the reaction was monitored via 1 H NMR analysis. Redox-Triggered Change of the Nanoparticle Size. Nanoparticles prepared with copolymer P1 at a concentration of 5.0 mg ml 1 were used for testing the redox response of the disulfide bonds. Three experiments at different concentrations of glutathione (GSH) and nanoparticles were carried out. In the first experiment, 0.20 ml of a stock solution of GSH (0.10 M) in 1.0 M PBS was added to 1.80 ml of a nanoparticle solution (C = 5.0 mg ml 1 ; C(S S bonds) = 1.4 mm), which resulted in a final GSH concentration of 10 mm. DLS measurements were recorded before and 10 min after the GSH was added. The mixture was extracted three times with chloroform. Then, the organic phase was dried and analyzed by SEC. A second experiment was performed by adding 0.20 ml of a stock solution of GSH (0.10 mm) in 1.0 M PBS to 1.8 ml of a nanoparticle solution (C = 5.0 mg ml 1 ; C of S S bonds = 1.4 mm), which resulted in a final GSH concentration of 10 μm. DLS measurements were recorded before and after the addition of GSH at different times (10, 120, and 240 min). During the third experiment, 0.20 ml of a stock solution of GSH (0.10 mm) in 1.0 M PBS was added to 1.8 ml of a nanoparticles 7054

4 solution (C = mg ml 1 ; C of S S bonds = 1.4 μm), which resulted in a final GSH concentration of 10 μm. DLS measurements were recorded before and after the addition of GSH at different times (10, 120, and 240 min). Nile Red Encapsulation and in Vitro Release Study. 1.8 mg of Nile red and 3.6 mg of lyophilized nanoparticles, prepared with copolymer P1 at concentration of 2.0 mg ml 1 (Table 3, run 2), were dissolved in 2 ml of DMF, and the solution was stirred for 18 h. Afterward, the solution was dialyzed against deionized water for 24 h. The total volume after dialysis was 12 ml. The size of the nanoparticles after Nile red encapsulation was determined by DLS. To quantify the Nile red loading, 1 ml of the solution was lyophilized and dissolved in 1 ml of MeOH. The concentration of encapsulated Nile red was measured by UV, knowing that in MeOH its maximum of absorbance is at 552 nm and the value of ε = mol 1 cm 1. The in vitro Nile red release was evaluated at two different concentrations of GSH. In the first experiment, 0.20 ml of a stock solution of GSH (0.10 M) in 1.0 M PBS was added to 1.80 ml of the nanoparticles solution, which resulted in a final GSH concentration of 10 mm. A second experiment was performed by adding 0.20 ml of a stock solution of GSH (0.10 mm) in 1.0 M PBS to 1.8 ml of a nanoparticles solution, which resulted in a final GSH concentration of 10 μm. Fluorescence measurements were recorded on the nanoparticles solution prepared with copolymer P1 at concentration of 2.0 mg ml 1 (Table 2, run 2), on the nanoparticles solution containing Nile red before and after the GSH was added. RESULTS AND DISCUSSION Copolymer Synthesis and Postpolymerization Modification. Functional PLA chains, having different chain compositions, were obtained by ring-opening copolymerization of LA and a previously reported functional monomer, TrtS- LA, 6 using SnOct 2 as the catalyst. The pendant trityl-s groups (TrtS) were then modified into pyridyl disulfide (PDS) to enable the reaction with HS-PEG-SH; hence, the nanoparticles were prepared (Scheme 1). Scheme 1. Synthetic Route for Disulfide Cross-Linked PEG PLA Nanoparticles high conversion for both monomers was achieved, the sample was collected in good yield, and the composition was evaluated by 1 H NMR, which reflected that of the feed. The SEC analysis in CHCl 3 indicated a monomodal molecular weight distribution with a molecular weight of 43.8 kda and a narrow dispersity, Đ = 1.2. A second run was performed after increasing the amount of TrtS-LA in the feed to 15 mol % (Table 1, sample P2a). A low conversion for both monomers was obtained after 42 h. The copolymer sample was obtained in low yield, 34%, with a low molecular weight M n = 8.1 kda. A copolymerization with the same feed ratio was carried out for a longer reaction time (66 h, sample P2b, Table 1), achieving a slightly higher conversion. However, the molecular weight value increased to 29.1 kda, and the dispersity value was broader, Đ = 1.4. All the copolymerizations were performed using as-received monomers and initiator; the difference in molecular weight may be due to presence of adventitious water, which can also act as initiator. The last run was performed with a feed ratio for LA/TrtS-LA of 70:30 (Table 1, sample P3). A higher conversion for both monomers, compared to sample P2b, was achieved after 66 h with good yield (68%). The molecular weight value of sample P3 was slightly lower than that of samples P1 and P2b, M n = 22.1 (Đ = 1.4). The conversion of TrtS-LA was generally higher than that of LA for all the copolymerizations. Consequently, the copolymer composition was enriched in TrtS-LA with respect to the feed. The NMR characterization was performed on all the samples similar to that reported in the literature. 6 It revealed that the functional units were randomly distributed, and consecutive TrtS-LA units were never incorporated along the polymeric chains. The effect of the copolymer composition on the thermal properties was studied by means of differential scanning calorimetry (DSC). The thermograms registered during the second heating scan of copolymers with different numbers of TrtS-LA units (Table 1, samples P1, P2b, and P3) are compared in Figure 1. All the copolymers were amorphous, and the glass transition temperature, T g, increased with the increasing the content of TrtS-LA. The T g for the copolymer was higher than that of the PLA. This observation could be derived from the stiffening of the polymer backbones caused by the bulky pendant thio-trityl Copolymerization of TrtS-LA with LA using SnOct 2 as the catalyst, and ethylene glycol as the cocatalyst was carried out by varying the comonomer feed ratio at 110 C, with a monomerto-catalyst ratio of 1000:1 and monomer-to-initiator ratio of 250:1 (Table 1). The copolymers were characterized by NMR, SEC, and DSC. The first copolymerization was performed with a LA:TrtS-LA ratio of 95:5 (Table 1, sample P1). After 24 h, a Figure 1. DSC thermograms (run II) of poly(trts-la-co-la) with TrtS-LA compositions of (i) 6 mol %, (Table 1, sample P1); (ii) 20 mol % (Table 1, sample P2b); and (iii) 32 mol % (Table 1, sample P3). 7055

5 groups. A similar effect due to the steric hindrance of the substituents on the T g of cyclohexyl-substituted polyglycolides has been reported. 20 Latent thiol functionalities, protected by trityl thioether and randomly distributed along the polymeric chains, were available for further modification, which could open the way for a wide range of possibilities for the fabrication of functional materials. Indeed, due to its versatility, thiol chemistry plays an important role in the synthesis, modification, and functionalization of polymeric materials. 7 In particular, polymers functionalized with pyridyl disulfide groups (PDS) can undergo a fast exchange reaction with free thiol groups to produce disulfide bonds 21,22 that may be cleaved in a reductive environment. 19 To achieve this, the pendant TrtS- groups were modified into PDS groups by a two-step procedure (Scheme 2), following a previously reported procedure. 6 Scheme 2. Modification of the Pendant TrtS Groups into Pyridyl Disulfide (PDS) Functionalities During the first step, the trityl protecting groups of the poly[(trts-la)-co-la] samples were cleaved by reacting with trifluoroacetic acid (TFA) and Et 3 SiH. Afterward, crude copolymers bearing free thiol functionalities, poly[(hs-la)- co-la] samples, were allowed to react with an excess of 2,2 - dipyridyl disulfide to obtain poly[(pds-la)-co-la] in high yield. The copolymers were characterized by NMR and SEC. The 1 H NMR spectra of the poly[(pds-la)-co-la] samples showed three different signals in the aromatic region due to the protons of the pyridyl ring. A shift in the methylene group, CH 2 S, with respect to the chemical shift of the native copolymer (from ppm to ppm) was also detected. Similar shifts have been observed for copolymers of TrtS-LA with ε-caprolactone, poly[(trts-la)-co-cl]. 6 The other signals of the main chain remained unaffected during the reactions, and the initial copolymer composition did not vary at all (Figure S3). SEC measurements indicated monomodal and narrow dispersity for samples P1 and P3, while for samples P2a and P2b a slight increase of M n and broader dispersities with respect to the native polymers were observed. The slight change in molecular weight and dispersity values with respect to the native sample may have been due to the performed modification (Table 1). However, the increase of M n and Đ observed for samples P2a and P2b may indicate the occurrence of side reactions during the postpolymerization modification; for instance, formation of S S bonds that may occur either in the presence of adventitious oxygen or by reaction of unreacted free thiol and pyridyl disulfide groups along the chains. Such reaction is of course more probable when the amount of functional groups is higher. As a result, a small shoulder in the SEC chromatogram was observed for samples P2a and P2b (Figures S10 and S11). Nonetheless, the postpolymerization modification was carried out with high yield and without affecting the polymer main chain, i.e., the degree of polymerization and the composition. Nanoparticles Preparation. The PDS pendant functionalities on the poly[(pds-la)-co-la] samples were exploited to induce a disulfide exchange reaction with telechelic HS-PEG- SH, with the aim to fabricate cross-linked nanoparticles. Because of the presence of the disulfide bonds, these nanoparticles should also be redox-responsive and selectively disaggregate in a reducing environment (i.e., by the presence of glutathione). One of the easiest procedures for micelle formation based on amphiphilic PLA PEG diblock copolymers involves precipitating an organic solution of copolymers in water phase, which is called nanoprecipitation. 23 Attempts to obtain the nanoparticles by nanoprecipitation techniques were performed first using THF as the organic solvent. When solutions of poly(pds-laco-la) and HS-PEG-SH in THF were poured simultaneously or consecutively in water, large aggregates were formed. In contrast, when the poly[(pds-la)-co-la] samples were mixed with the telechelic HS-PEG-SH in dimethylformamide (DMF) and the organic solution was dialyzed against water, particles with nanoscopic dimensions were formed. The nanoparticles were prepared by adding a solution of the HS-PEG-SH in DMF to a solution of the chosen copolymer. The final total concentration of both polymers in DMF was 10 mg ml 1, with equimolar concentrations of SH and PDS groups. The reaction was carried out for all the copolymers, and the nanoparticle dimensions were evaluated by dynamic light scattering (DLS). The data are summarized in Table 2. The DLS analysis of the obtained nanoparticles was carried out in DMF (Table 2), and it indicated that the reaction resulted in the formation of nanoparticles having dimensions smaller than 200 nm. However, the size values estimated in DMF should be considered with special care. The size distribution was broad for almost all cases, with PDI values close to 1.0 (Table 2). Moreover, no correlation between the number of functional groups, the molecular weight, and the nanoparticles size determined in DMF was observed. The nanoparticles solutions obtained in DMF were dialyzed against deionized water for 24 h, using a membrane with a molecular weight cutoff between 100 and 500 Da. DLS measurements were then performed in water; the data are summarized in Table 2. Contrary to the data obtained in DMF, the size of the nanoparticles was well controlled in water. Narrower size distributions were observed, and the average values determined for the three measurement were much closer to each other. Interestingly, the sizes of the particles estimated in water were generally larger than those measured in DMF. Moreover, in water, the dependence of the size values on the number of functional groups as well as on the molecular weight was recognized. When the content of functional units was 20 mol %, the size increased from 167 to 300 nm by increasing the molecular weight of the native copolymer (Table 2, samples P2a and P2b). For sample P1, which has a lower number of functional units but a higher molecular weight, particles with a size of 180 nm were obtained (Table 2). For sample P3, which contains the highest number of functional units (32 mol %), the mean diameter of 254 nm can be attributed to the lower molecular weight, 18.6 kda, of the native copolymer (Table 2). Finally, nanoparticles prepared with sample P3 showed a broad size distribution in water. Study of the Reaction between HS-PEG-SH and Poly(PDS- LA-co-LA). To gain a deeper insight into the formation of the 7056

6 nanoparticles, the reaction between the functionalized polymer and HS-PEG-SH was monitored by UV and NMR spectroscopy. The driving force of the disulfide exchange reaction is the release of 2-pyridinethione, which enabled easy and quantitative monitoring of the reaction. The 2-pyridinethione concentration in the reaction mixture was estimated by UV spectroscopy, using the known molar extinction coefficient at 375 nm (ε = mol 1 cm 1 ) in DMF. 24 The UV spectra of the samples prepared with the copolymer P2a (Table 2) are reported in Figure 2. Figure 2. UV spectra for the reaction between P2a and HS-PEG-SH (Table 2), showing the formation of 2-pyridinethione (λ = 375 nm) after 30 min. The absorption band of the 2-pyridinethione (λ max = 375 nm) is clearly evident in the spectrum of the reaction mixture after 30 min (Figure 2). The total release of the 2- pyridinethione was generally achieved after 30 min. However, the reaction was allowed to stir for 24 h to ensure full conversion of the PDS groups into -S-S-PEG-. In these conditions, a graf ted and cross-linked PLA PEG network should have been obtained by the disulfide exchange reaction between the PDS groups along the PLA chain and the HS end groups of the PEG. Therefore, the binding of the telechelic HS-PEG-SH branches to the PLA chains should occur first, and then, the formed cross-linked species should be assembled into a nanoparticle with a hydrophobic core and hydrophilic shell when it is dissolved in the water phase. The reaction was also followed in situ using a 1 H NMR analysis in CDCl 3 for sample P2a (Figure 3). For comparison, the 1 H NMR spectra of the unreacted polymers, i.e., HS-PEG- SH and copolymer P2a, are also reported in Figures 3ii. However, 1 H NMR could not allow an exact estimation of the released 2-pyridinethione during the reaction progress because its signals overlap with the signals of the PDS groups along the polymeric chains. The three main signals at 3.6 ppm (a), 2.7 ppm (b), and 1.6 ppm (c) in the 1 H NMR spectrum of the HS-PEG-SH (Figure 3i) were attributed respectively to the CH 2 O, CH 2 S, and SH moieties. The integral ratio was consistent with the presence of only the HS-PEG-SH species; no oxidation of the thiol groups to disulfides had occurred. HS-PEG-SH and poly[(pds-la)-co-la] were dissolved in CDCl 3, with equimolar amounts of SH and PDS groups, and the final concentration of both polymers was 10 mg ml 1.A 1 H NMR spectrum was recorded after 24 h (Figure 3iii). Interestingly, the intensity of the signal at 2.7 ppm ( CH 2 S, b) decreased, and a new signal appeared at 2.9 ppm ca. (b ). Moreover, a change in the signal at 3.25 ppm (PLA CH 2 S, g) was also Figure 3. Selected regions of 1 H NMR spectra (400.0 MHz, CDCl 3 ) of HS-PEG-SH (i); poly(pds-la-co-la), sample P2a (ii); the reaction mixture after 24 h (iii); and the reaction mixture after 48 h (iv). : methanol. observed. After 48 h (Figure 3iv), the signal at 2.7 ppm (b) completely disappeared, and the intensity of the signal at 2.9 ppm increased (b ). The peaks of the pyridyl moiety (h, i, j, k) in the aromatic region also decreased over time (Figures 3iii and 3iv). Thus, the presence of the 2-pyridinethione band in the UV spectrum, the decreased intensity of the aromatic signals, and the shift of the signal of the CH 2 S of the PEG block from 2.7 ppm (b) to 2.9 ppm (b ) in the 1 H NMR spectra of the reaction mixture indicated that the disulfide exchange reaction between poly[(pds-la)-co-la] and HS- PEG-SH occurred. If the UV and NMR data revealed that the reaction occurred, then the DLS data showed that it led to the formation of nanoparticles with sizes in the range nm. Effect of Concentration on Nanoparticles Size. For biomedical applications, particularly for drug delivery systems, the dimensions of nanoparticles should be smaller than 200 nm to avoid fast clearance and to achieve the preferred biodistribution. 25 As a result, the nanoparticles obtained with copolymers P1 and P2a, having the smallest dimensions in water and the narrowest size distribution (Table 2), are the most promising and were selected for further evaluation. To assess the effect of the concentration on the nanoparticles size, a first set of screenings was conducted with copolymer P1, having 6 mol % of PDS-LA functional units. The nanoparticles were prepared in DMF at different concentrations and then dialyzed against deionized water, as previously described. The results obtained by DLS are summarized in Table 3 (runs 1 5). The nanoparticles distributions were quite broad in DMF, while narrower size distributions were revealed for nanoparticles in water after dialysis. 7057

7 For copolymer P1, the nanoparticles dimensions did not vary with the concentration up to 10.0 mg ml 1. The estimated size was in the range nm in DMF and nm in water (Table 3, runs 1 4). The dimensions of the particles were well controlled in the water phase for concentrations in the range mg ml 1, with a narrow size distribution (Table 3, runs 2 4). At concentrations as high as 20.0 mg ml 1, bigger particles with a size of approximately 3000 nm were obtained in DMF, which precipitated in water after dialysis, thus hampering the DLS analysis (Table 3, run 5). Moreover, the diameter values obtained in water for the nanoparticles prepared at a lower concentration, 0.5 mg ml 1, diverged from the values recorded in DMF, resulting in larger nanoparticles in the aqueous phase (Table 3, runs 1) than the ones obtained at higher concentrations (Table 3, runs 2 4). Most likely, at lower concentrations, intrachain cross-links occurred, and on the contrary, the probability of interchain cross-links was higher when the polymer concentration increased. When particles with a higher number of interchain cross-links swelled in the water phase, they might have assembled into smaller sizes since more PLA blocks were trapped in the core; thus, the hydrophobicity increased, and as a consequence, the resulting nanoparticle cohesion was higher. Similar observations have been reported for PEG PLA micelles. 11,12 The same screening while varying the concentration was carried out with sample P2a, which has a molecular weight of 10.3 kda and 20 mol % functional PDS-LA units. The results obtained by DLS are also summarized in Table 3. A plot of the nanoparticle sizes as a function of the concentration is shown in Figure 4. for samples prepared at low concentrations (Table 3, runs 6 8), while at concentrations equal to or higher than 10 mg ml 1 a good agreement between the values estimated in DMF and in water was observed (Table 3, runs 9 11), and the particles sizes slightly increased by increasing the concentration (Figure 4). Behavior and Stability of the Nanoparticles in Water. The DLS analysis (Tables 2 and 3) described in the above section showed that the size values of the particles estimated in water were generally higher than the values obtained in DMF. Moreover, the nanoparticle sizes were better controlled in water than in DMF. The average size values determined using the three measurements were much closer to each other in water than in DMF, and the PDI values were narrower. The huge gap between the three different measurements and the broader PDI values in DMF could indicate either that the particles sizes varied in a wide range or that the particles shape was not spherical; therefore. the light was scattered differently because of the anisotropic shape. Indeed, the two blocks, i.e., PLA and PEG, were both soluble in DMF, and the nanoparticles should not have self-assembled in a specific manner. On the other hand, the better distribution of the particles sizes evaluated in water could be due to the swelling of the PEG blocks which occupy the outer shell in the water phase. Our hypothesis is that the nanoparticles could assemble into spherical shape with flowerlike structures having a PLA core and a swollen shell made of PEG loops (Figure 5). Figure 4. Plot of the size as a function of the concentration obtained by DLS for the nanoparticles prepared with sample P2a in DMF and after dialysis in water. The same consideration of the results obtained with sample P1 also applied for the data obtained with sample P2a. Thus, nanoparticles with sizes in the range nm were obtained in DMF with a large size dispersion. The particle size values were approximately constant for the concentrations of mg ml 1 (Table 3, runs 6 9). A size increment was observed by increasing the total concentration of the copolymers to 20 and 40 mg ml 1 (Table 3, runs 9 11). At higher concentrations, the interchain cross-links became more probable, thus provoking the increase of the particle sizes. The sizes values estimated in water were higher than the values obtained in DMF. Additionally, in this case, the sizes estimated in water diverged from the values recorded in DMF Figure 5. Nanoparticles assembly in DMF and in water. 1 H NMR spectra, in different solvents, were recorded to support this hypothesis. The nanoparticles obtained with copolymer P2a at a concentration of 10 mg ml 1 (Table 3, run 9) were freeze-dried and then analyzed in DMF-d 7 and D 2 O(Figure 6). The spectrum obtained in DMF-d 7 revealed the signals of both the PLA and the PEG blocks. In contrast, the spectrum obtained in D 2 O revealed only the PEG blocks. These results clearly indicate that the PLA block behaved as the inner core in the nanoparticles, and it was hidden by the PEG, which constituted the outer shell in the water phase. An analogous structure was reported for photo-cross-linked nanogels composed of PLA PEG PLA triblock copolymers. 26 The freeze-dried nanoparticles were also analyzed by SEM. In Figure 7, the SEM images of the freeze-dried nanoparticles 7058

8 Macromolecules 1 to 162 ± 1 nm, and the same dimensions were measured up to 100 times dilution, i.e., C = 0.05 mg ml 1. At higher dilutions, 1000 times and C = 5.0 μg ml 1, the DLS results indicated that the nanoparticles sizes were slightly larger; however, this result could be due to the higher attenuation parameter detected at lower concentrations. To verify the redox response properties of the prepared nanoparticles containing disulfide bonds, we evaluated how the GSH induced structural changes. For this purpose, experiments with different concentrations of GSH were carried out. The first experiment was performed using a GSH concentration of 10.0 mm, i.e., the concentration of GSH that can be found inside cells. Ten minutes after the GSH was added to the nanoparticles solution, the DLS analysis indicated the formation of large aggregates with sizes of approximately 3.0 μm, and after 20 min, a precipitate was observed. These results indicate that in these conditions the cleavage of the disulfide bonds occurred, and the PLA blocks, no longer linked to the PEG, started to form large aggregates and then precipitated since they were not soluble in the water phase. The mixture was extracted in chloroform, and the SEC analysis of the organic phase revealed a peak with an Mn of 46.4 kda. This value is close to the Mn of sample P1 (Table 1). Therefore, with cellular concentrations of GSH, the nanoparticles showed redoxresponsive disruption of their structures. When the concentration of GSH was 10 μm, which is the concentration of GSH that can be found outside of cells, DLS measurements carried out at different times (10, 120, and 240 min) for an incubated solution at 37 C revealed that the size did not change (Table S2), and the nanoparticles were stable in the presence of this concentration of GSH. In this condition, however, the disulfide bonds were in large excess with respect to the GSH. Therefore, a third experiment was carried out at a GSH concentration of 10 μm, and the nanoparticle solution was also diluted to a final concentration of mg ml 1 (C of S S bonds = 1.4 μm) in order to have an excess of GSH. The solution was incubated at 37 C; DLS measurements carried out at different times revealed that the size did not change (Table S3), and the nanoparticles were stable in the presence of this concentration of GSH. In Vitro Tests of Loading and Release of Nile Red. In vitro tests of drug loading and controlled release were performed by fluorescence measurements using Nile red as a model guest molecule. Nile red is a hydrophobic fluorescent dye poorly water-soluble. If it is encapsulated in the hydrophobic environment of a micelle core, its fluorescence can be detected in water phase, while the fluorescence dramatically decreases when it is released from the micelles.27 The Nile red was loaded into pre-formed nanoparticles (Table 3, run 2) by dissolving the hydrophobic guest and lyophilized nanoparticles in DMF, with a weight ratio Nile red to nanoparticles of 1:2. After stirring for 18 h, the solution was dialyzed against deionized water, resulting in a light magenta colored solution. The diameter of the nanoparticles after Nile red loading, estimated by DLS, resulted to be 215 ± 1 nm, larger than the diameter measured for the native nanoparticles (194 ± 1 nm; Table 3, run 2), with slightly increased PDI from 0.10 (Table 3, run 2) to To estimate the Nile red loading, the absorbance at 552 nm of a lyophilized nanoparticles dissolved in MeOH was measured by UV. The concentration of Nile red resulted to be M, which corresponds to 11 μg of encapsulated Figure 6. 1H NMR spectra of freeze-dried nanoparticles prepared at a concentration of 10 mg ml 1 (Table 2, run 2) in DMF-d7 (i) and D2O (ii). (*)Deuterated solvent residual peaks. prepared with the copolymer P1 containing 6 mol % PDS-LA units at a concentration of 10.0 mg ml 1 are reported (Table 1, run 1). Figure 7. SEM images of freeze-dried nanoparticles prepared with copolymer P1 at a concentration of 10.0 mg ml 1 (Table 3, run 9) at a magnification of 40K (i, ii) and 100K (iii, iv). The SEM images nicely show that spherical and discrete particles were obtained in the water phase. The particles sizes were in the range nm, which is slightly lower than the range estimated by DLS (Table 3, run 9). This could be a consequence of the drying process. During the drying step, the particles may have shrunk, leading to a decreasing of their diameter. The stability of the nanoparticles in water over time and after dilution was evaluated for the samples prepared in run 3 (Table 3), which resulted in particles with the smallest size. A DLS analysis was performed after 35 days. Then, the nanoparticle solution was also diluted several times, and the size was estimated by DLS (Table S1). The size of the nanoparticles was not affected by the dilution. The diameters ranged from 159 ± 7059

9 Figure 8. Fluorescence emission spectra of nanoparticles solution (Table 3, run 3) and nanoparticles solution containing Nile red prior and after the addition of GSH 10 μm (i) and GSH 10 mm (ii) at 2, 4, and 24 h. Nile red or to a transport efficiency (μg dye/mg nanoparticles) of 3 μg mg 1. The Nile red release was evaluated at cellular (10 mm) and extracellular concentration (10 μm) of GSH. Fluorescence measurements of the nanoparticles containing Nile red and after 2, 4, and 24 h that the GSH was added were conducted (Figure 8). Fluorescence spectra of the native nanoparticles solution were also recorded as blank. When an extracellular concentration of GSH (10 μm in PBS buffer) was added to the nanoparticles solution containing Nile red, no loss in the fluorescence intensity was observed up to 24 h(figure 8i). The slightly increase of the intensity observed when the GSH at 10 μμ was added was probably due to changing in the solution. On the other hand, a profound loss in fluorescence was recorded in the presence of cellular concentration of GSH (Figure 8ii). In this case a precipitate, probably constituted by PLA blocks and Nile red, was clearly observed, while the solution became colorless (Figure S13). These data clearly indicated that the S S bonds were reduced, the nanoparticles were disassembled, and the Nile red was released. Similar observations were described for Nile red encapsulated nanoparticles made of functional poly- (acrylamide)s. 28 The whole picture demonstrated that the nanoparticles are good nanocarriers for hydrophobic guest molecules. They were stable and retained the Nile red up to 24 h in extracellular conditions, while, thanks to redox-responsive behavior of the disulfide cross-links, they allowed the Nile red release upon the intracellular reductive environment. CONCLUSIONS Cross-linked polymeric nanoparticles have great potential to be used as drug carriers since they avoid the dilution problems that micellar systems have when injected in the body. The introduction of dynamic cross-links in the polymeric network can enable the controlled release of the drug under specific stimuli. We prepared cross-linked polymeric nanoparticles by a disulfide exchange reaction between functionalized PLAs and telechelic HS-PEG-SH. Ring-opening copolymerization of the TrtS-LA with LA using stannous octanoate was carried out for the first time, and it enabled the synthesis of polymers bearing latent thiol functionalities with a monomodal molecular weight distribution and narrow dispersity. Four different copolymers were prepared with different numbers of functional groups and/or different molecular weights. Afterward, the functional groups were transformed to pyridyl disulfide without affecting the main polymer chain. Polymeric networks composed of PLA and PEG blocks linked by disulfide bonds were prepared by a disulfide exchange reaction in DMF between the functionalized PLAs and telechelic HS-PEG-SH. They assembled into discrete nanoparticles with a hydrophobic core and hydrophilic shell with sizes in the range of nm in water. When copolymers with low molecular weights or low numbers of functional groups were used, nanoparticles with diameters smaller than 200 nm were prepared, which are suitable to be used as drug carriers. Moreover, their size could be modulated by changing the polymer concentration. Notably, PEG arms with a molecular weight 1.0 kda were sufficient for shielding the PLA core and enabling the solubility of the nanoparticles in water, even when only 6 mol % of functional units were incorporated in the copolymer. This is an important result because PEG with a low molecular weight can be excreted from the body within short times. Notably, the prepared nanoparticles had great stability over time and after dilution. This ensure the stability of the carrier when it is injected into the body. Moreover, they were stimuliresponsive to the GSH concentration: they disassembled quickly in the presence of a redox environment, such as cellular concentrations of GSH, where the disulfide bonds were easily reduced. In contrast, the nanoparticles were stable at extracellular concentrations of GSH. This redox-responsiveness enables the possibility to target the release of a drug inside of cells. This was further assessed by encapsulating a hydrophobic guest molecule, the Nile red, in preformed nanoparticles and showing that the release occurred only at cellular concentration of GSH. Therefore, we believe that the prepared nanoparticles hold great potential as drug delivery systems, and thanks to the specific redox-responsive behavior, they may allow for controlled and targeted drug release. 7060