Article pubs.acs.org/Biomac
Polymersomes from Dual Responsive Block Copolymers: Drug Encapsulation by Heating and Acid-Triggered Release Zeng-Ying Qiao, Ran Ji, Xiao-Nan Huang, Fu-Sheng Du,* Rui Zhang, De-Hai Liang, and Zi-Chen Li* Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Department of Polymer Science & Engineering, College of Chemistry & Molecular Engineering, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: A series of well-defined thermoresponsive diblock copolymers (PEO45-b-PtNEAn, n = 22, 44, 63, 91, 172) were prepared by the atom transfer radical polymerization of trans-N-(2ethoxy-1,3-dioxan-5-yl) acrylamide (tNEA) using a poly(ethylene oxide) (PEO45) macroinitiator. All copolymers are water-soluble at low temperature, but upon quickly heating to 37 °C, laser light scattering (LLS) and transmission electron microscopy (TEM) characterizations indicate that these copolymers self-assemble into aggregates with different morphologies depending on the chain length of PtNEA and the polymer concentration; the morphologies gradually evolved from spherical solid nanoparticles to a polymersome as the degree of polymerization (“n”) of PtNEA block increased from 22 to 172, with the formation of clusters with rod-like structure at the intermediate PtNEA length. Both the spherical nanoparticle and the polymersome are stable at physiological pH but susceptible to the mildly acidic medium. Acid-triggered hydrolysis behaviors of the aggregates were investigated by LLS, Nile red fluorescence, TEM, and 1H NMR spectroscopy. The results revealed that the spherical nanoparticles formed from PEO45-bPtNEA44 dissociated faster than the polymersomes of PEO45-b-PtNEA172, and both aggregates showed an enhanced hydrolysis under acidic conditions. Both the spherical nanoparticle and polymersome are able to efficiently load the hydrophobic doxorubicin (DOX), and water-soluble fluorescein isothiocyanate-lysozyme (FITC-Lys) can be conveniently encapsulated into the polymersome without using any organic solvent. Moreover, FITC-Lys and DOX could be coloaded in the polymersome. The drugs loaded either in the polymersome or in the spherical nanoparticle could be released by acid triggering. Finally, the DOXloaded assemblies display concentration-dependent cytotoxicity to HepG2 cells, while the copolymers themselves are nontoxic.
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INTRODUCTION In recent years, polymeric vesicles, also called polymersomes, have attracted growing interest due to their potential applications in various fields such as drug/gene delivery, nanoreactors, artificial organelles, and bioimaging.1−4 As drug carriers, polymersomes have many advantages over the classical lipid liposomes, including adequate stability, long circulation in bloodstream, good mechanical property, and great potential for advanced chemical functionalization.5−8 In general, the lesser membrane permeability of a polymersome is helpful to reduce the premature leakage of the payload; however, it inhibits the rapid drug release at the desired sites and may diminish the therapeutic efficacy. Thus, numerous sensitive polymersomes that respond to various stimuli have been developed as potential carriers for intelligent drug/gene delivery.9−18 Convenient and/or efficient drug loading, in particular for water-soluble biomacromolecules, and the temporally and/or spatially programmed drug release represent two of the potential benefits of these responsive polymersomes used as the vehicles of therapeutic agents. In comparison with polymeric micelles or other solid spherical nanoparticles that are usually used as the vehicles of © XXXX American Chemical Society
hydrophobic drugs, polymersomes have hydrophilic reservoirs as well as relatively thick hydrophobic membranes; they can thus encapsulate both water-soluble bioactive agents and hydrophobic drugs for combinatorial therapeutics.6,7,11 Conventional preparation of most amphiphilic copolymer polymersomes requires harsh conditions, including the use of organic solvent, sonication, and/or time-consuming processes, which may denature the fragile payloads such as proteins. To overcome these drawbacks, in the past several years, various stimuli-responsive polymersomes have been prepared from the double hydrophilic block copolymers or copolypeptides by simply changing temperature19−23 or pH24−27 of the media, and hydrophilic compounds such as doxorubicin hydrochloride (DOX·HCl),28−30 DNA,31 or proteins32 can be encapsulated during the polymersome formation. In another approach, polyion complex vesicles with tunable size and pH-dependent membrane permeability have been fabricated through a facile Received: February 5, 2013 Revised: March 28, 2013
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proceduresimple mixing of two water-soluble and oppositely charged block copolymers.33 Programmed drug release is a key issue for a drug delivery system.34 Various stimuli including temperature,28,35−37 pH,38−41 redox potential,20,42,43 light,44,45 ultrasound,46 biomolecules,47,48 and so forth, have been used to tune the drug release from polymersomes. In particular, pH-responsive polymersomes have been studied most extensively because of the existence of numerous pH gradients in both normal and pathological states.49−52 While most of the pH-responsive polymersomes were derived from polymers containing weakly acidic or basic groups, recently, a new type of degradable polymersome formed by the diblock copolymer of poly(ethylene oxide) (PEO) and polycarbonate with pendent acetals was reported. The polymersome could simultaneously load paclitaxel and DOX·HCl at pH 7.4 by a solvent-switch technique, and quick release of the payloads in acidic media was observed.53 Recently, we developed a family of acid-labile thermoresponsive polymers whose lower critical solution temperatures increased with the hydrolysis of the pendent ortho ester groups.54−56 Furthermore, we prepared a series of acid-labile thermoresponsive block copolymers composed of a PEO5000 (PEO114) block and a PtNEA block, and found they could form micelle-like nanoparticles by directly heating the copolymer solution above the critical aggregation temperature (CAT). However, these nanoparticles can only encapsulate the hydrophobic compounds.57 We reasoned that a polymersome can be formed in a similar way by increasing the relative chain length of PtNEA block to PEO. In this paper, another series of diblock copolymers containing a short PEO segment and PtNEA blocks with different lengths were designed and synthesized. Upon heating above their CATs, these copolymers spontaneously formed polymersomes or spherical solid nanoparticles, depending on the length of the PtNEA block. Watersoluble biomacromolecules can be encapsulated in the polymersomes simply by heating of the polymer aqueous solution without using any organic solvent. By contrast, hydrophobic compounds are expected to be loaded into both polymersomes and spherical nanoparticles. These drug-loaded vehicles are stable at physiological pH but gradually disassembled in mildly acidic medium, triggering the payload release (Scheme 1). We speculate that these acid-labile copolymers are promising candidates as the carriers of multidrugs for site-specific delivery.
Article
EXPERIMENTAL SECTION
Materials. Nile red (NR, Aldrich), DOX·HCl (Beijing Huafeng United Technology Co.), fluorescein isothiocyanate (FITC, Aldrich), and lysozyme (TCI) were used as received. FITC-lysozyme (FITCLys) was synthesized by the reaction of FITC and lysozyme (molar ratio: FITC/lysozyme = 2) in sodium acetate buffer (pH 9.0) for 15 h at ambient temperature in dark, followed by dialysis in phosphate buffered saline (PBS) solution (pH 7.4) at 10 °C for 3 days in the dark and lyophilization. The block copolymers (PEO45-b-PtNEA22−172, P1P5) with different PtNEA lengths were synthesized by atom transfer radical polymerization (ATRP) following the published protocol.57 Other solvents and reagents were used as received. Laser Light Scattering (LLS). The dynamic light scattering (DLS) and static light scattering (SLS) measurements were performed on a commercial spectrometer (Brookhaven Inc., Holtsville, NY) composed of a BI-200SM goniometer, a BI-TurboCorr digital correlator, and a vertically polarized solid state laser source (100 mW, 532 nm, CNI, Changchun, China). The hydrodynamic radius (Rh) and z-averaged root-mean-square radius of gyration (Rg) were obtained from the DLS and SLS measurements, respectively.57 Before the LLS measurement, the copolymer solution (0.02 mg/mL for P2−P5, or 0.2 mg/mL for P1) in 10 mM pH 8.4 phosphate buffer (PB) was filtered into a dustfree vial through a Millipore 0.22 μm PVDF membrane at 4 °C, and quickly heated to 37 °C in 5 min and maintained at this temperature for an additional ∼2 h. To study the effect of initial concentration on the aggregation, the aggregate dispersions with a polymer concentration of 2.0 mg/mL were also prepared by the same procedure. The relatively concentrated dispersions of copolymers P2−P5 were diluted to 0.02 mg/mL by adding 20 μL of the aggregate dispersion into 2 mL of filtered PB at 37 °C, and the diluted dispersions were incubated at 37 °C for another ∼2 h prior to the LLS measurements. For the pHdependent hydrolysis experiments, the copolymer aggregate dispersions (initial concentration 0.02 mg/mL) were prepared by following the aforementioned protocol, and the obtained LLS data at 37 °C were used as those for 0 min time point. Then, the pH of the dispersions was adjusted by adding 5.0 M pH 4.6 (or 5.0) acetate buffer, and the LLS measurements were performed at the desired time points. The final buffer concentration was 50 mM. Transmission Electron Microscopy (TEM). The morphologies of the copolymer aggregates were observed by TEM (JEOL JEM100CXII) with an acceleration voltage of 100 kV. The copolymer aggregate dispersions (2.0, 0.2, or 0.05 mg/mL) in 10 mM pH 8.4 PB were formed by the same procedure as that used for the LLS measurements. TEM samples were prepared by placing one drop of the aggregate dispersions (pH 8.4) on a copper mesh. After 30 s, most of the liquid was removed by blotting with a filter paper, and then the remnant on copper mesh was air-dried at ∼40 °C in 5 min, followed by staining with uranyl acetate solution for 1 min. For the acidtriggered hydrolysis experiments, the aggregate dispersions were maintained at 37 °C, and then 5.0 M pH 4.6 (or 5.0) acetate buffer was added. At different time points, one drop of the dispersion was placed on a copper mesh and treated following the aforementioned procedure. NR Fluorescence. NR was used as a fluorescent probe to study the pH-dependent hydrolysis behavior of the copolymer aggregates. NR (20 μL ethanol solution, 1.0 × 10−3 mol/L) was added into 10 mL of copolymer solution (0.5 mg/mL in 10 mM pH 8.4 PB). The solution was quickly heated to and maintained at 37 °C overnight with stirring. After the NR-containing dispersion was adjusted to a desired pH by adding different buffers (PB for pH 7.4, 200 mM, or acetate for pH 4.6 and 5.0, 5.0 M), the fluorescence of the dispersion was measured at specific times on an F-4500 fluorometer (Hitachi) at an excitation wavelength of 545 nm. Loading and Release of FITC-Lys. FITC-Lys (2.0 mg) was added into 2.0 mL of copolymer P5 solution (5.0 mg/mL in 10 mM PB, pH 8.4) and kept at ∼4 °C for 48 h. This solution were quickly heated to 37 °C in ∼5 min and maintained at 37 °C under stirring for another 4 h, followed by dialysis against PB (pH 8.4, 10 mM) for 16 h at 37 °C (MWCO: 100 KDa). The loading capacity and efficiency
Scheme 1. Thermally Induced Formation and AcidTriggered Dissociation of Spherical Nanoparticles and Polymersomes
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Table 1. Characterization of PEO45-b-PtNEA Aggregates above CATa polymer P1 P2 P3 P4 P5
PEO45-b-PtNEA22 PEO45-b-PtNEA44 PEO45-b-PtNEA63 PEO45-b-PtNEA91 PEO45-b-PtNEA172
WPEOb (%) 31.2 18.5 12.7 9.8 5.5
Rh1c (nm) e
112 115 110 109 99.2
Rg1c (nm)
Rg1/Rh1
e
77.5 81.6 143 122 95.2
0.69 0.71 1.30 1.12 0.96
Rh2d (nm) f
122 228 280 245 193
CACg (mg/mL)
morphology
0.091 0.010 0.009 0.009 0.009
spherical NPh spherical NPh cluster polymersome polymersome
a All measurements were carried out at 37 °C. bWeight fraction of PEO in the block copolymers based on Mn,NMR in Table S1. cPrepared and measured at the polymer concentration of 0.02 mg/mL. dPrepared at 2.0 mg/mL, and diluted to 0.02 mg/mL at 37 °C before DLS measurements. e Prepared and measured at 0.2 mg/mL. fPrepared and measured at 2.0 mg/mL. gMeasured by fluorescence method using pyrene as a probe at 37 °C. hSpherical nanoparticle.
were determined by fluorescence method (Hitachi F4500, excitation wavelength: 495 nm). Briefly, 0.5 mL of the dialyzed aggregate dispersion was acidified by adding 10 μL of acetate buffer (pH 5.0, 2.0 M) and stirred at room temperature for ∼24 h. Then, this solution was neutralized by adding 0.5 mL of PB (200 mM, pH 7.4) and diluted to an appropriate volume by adding 50 mM PB (pH 7.4) to obtain the reliable fluorescence data. A series of solutions with different FITC-Lys concentrations in pH 7.4 PB (50 mM) were used for calibration (Figure S1). The measurements were performed in triplicate in the dark. For copolymer P2, a similar procedure was applied. The release profiles of FITC-Lys from the P5 polymersome were studied at 37 °C in the buffers of different pHs. The FITC-Lys loaded polymersome dispersion (3.0 mL, in 10 mM pH 8.4 PB) was placed into a dialysis tubing (MWCO: 100 KDa) and was immersed into 20 mL of buffer (PB for pH 7.4 or acetate for pH 5.0, 50 mM) under stirring at 37 °C. At the desired time points, 0.5 mL of the buffer was taken out and replenished with the same volume of fresh buffer. The solution taken out was adjusted to pH 7.4 before the fluorescence measurement. All the measurements were performed in triplicate in the dark. Loading and Release of DOX. Both the P2 nanoparticle and P5 polymersome, prepared by the fast heating protocol as aforementioned, can load hydrophobic anticancer drug DOX. DOX·HCl was first dissolved in dimethyl sulfoxide (DMSO; 4.0 mg/mL) or CH2Cl2 (2.0 mg/mL) in the presence of excessive triethylamine (DOX/TEA in molar ratio: ∼1/10) to afford free DOX solution, which was added to the aggregate dispersions in PB (2.0 mg/mL in PB, pH 8.4, 10 mM). After being stirred for 12 h at 37 °C, the dispersions were transferred into a dialysis tubing (MWCO: 10 KDa) and dialyzed against PB (10 mM, pH 8.4) at 37 °C for 16 h in the dark. During this period, the dialysis medium was changed thrice. To determine the loading capacity and efficiency, 1.0 mL of the dialyzed aggregate dispersion was diluted to 2.0 mL by adding 40 μL of pH 5.0 acetate buffer (5.0 M) and ∼0.9 mL of water before UV measurement. After stirring for 24 h, the copolymers were hydrolyzed thoroughly, and the DOX content in this solution was determined on a Shimadzu 2101 ultraviolet−visible (UV−vis) spectrometer at 485 nm. The amount of DOX loaded in the nanoparticles can be calculated according to the total volume of the dialyzed particle dispersions. The calibration curve was obtained by a series of solutions with different DOX concentrations in acetate buffer with the same pH (Figure S2, Supporting Information). The measurements were performed in triplicate in the dark. Loading capacity and loading efficiency (%) were defined as DOX in copolymer aggregate/copolymer aggregate (wt %) and DOX in copolymer aggregate/DOX in feed (wt %), respectively. Release experiments of DOX from the copolymer aggregates were carried out by a dialysis method. Briefly, 3.0 mL of the DOX-loaded aggregate dispersion (polymer concentration: 2.0 mg/mL) was quickly transferred into a dialysis tubing (MWCO: 10 KDa), which was immersed in 10 mL of buffers (PB for pH 7.4 and acetate for pH 5.0, 50 mM) with stirring at 37 °C. At a specific time interval, 1.0 mL of the dialysis medium was taken out for UV−vis measurement (485 nm) and replenished with the same volume of fresh medium. All the measurements were performed in triplicate in the dark. Simultaneous Loading of DOX and FITC-Lys. FITC-Lys (2.0 mg) was added into the P5 copolymer solution (2.0 mL, 5.0 mg/mL
in 10 mM pH 8.4 PB), which was kept at ∼4 °C for 48 h, and then heated to and maintained at 37 °C for 2 h. Then, DOX (0.8 mg in 0.2 mL DMSO) was added into the polymersome dispersion, which was stirred for 12 h at 37 °C. This mixed dispersion was transferred into dialysis tubing (MWCO: 100 KDa) and dialyzed against 10 mM pH 8.4 PB at 37 °C for 16 h under stirring in the dark. In order to measure the loading efficiency of DOX, the dual drug-loaded polymersome dispersion was transferred into another dialysis tube (MWCO: 3500 Da), which was immersed in 6.0 mL of acetate buffer (50 mM, pH 5.0) with stirring at 37 °C for 48 h. The dialysis media were replenished thrice and mixed together for UV−vis measurement to determine the amount of DOX loaded. The remained solution in the dialysis tubing was further dialyzed against 2 L of PB solution (pH 7.4) for 24 h at room temperature to remove the residual DOX, and analyzed by the fluorescence method to obtain the FITC-Lys loading capacity and efficiency. In Vitro Cytotoxicity Assay. Free P5 polymersome, the DOXloaded P2 and P5 nanoparticles (run 1 and run 4 in Table 2) were dispersed in phosphate buffer (10 mM, pH 7.4) at 37 °C. DOX was dissolved in DMSO and diluted into DOX aqueous solution of specific concentration. Linear PEO 2 KDa was used as a negative control. MTT assay was applied to evaluate the cytotoxicity of the samples in HepG2 cells. HepG2 cells were seeded in the 96-well plates and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. The aggregate dispersions or PEO solution with various concentrations (10 μL) were added to each well, and the cells were subjected to MTT assay after being incubated for another 24 h. The absorbance of the solution was measured on a Bio-Rad model 550 microplate reader at 570 nm. Cell viability (%) was equal to (Asample/Acontrol) x 100, where Asample and Acontrol denote the absorbance of the sample well and control well (without copolymer), respectively. Experiments were performed in triplicate.
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RESULTS AND DISCUSSION A family of thermoresponsive block copolymers (PEO45-bPtNEA) with different molecular weights were synthesized by ATRP using a PEO-macroinitiator (DP = 45) following our published procedure.57 DP of the PtNEA block (ranging from 22 to 172) was tuned by changing the feed molar ratio of monomer to macroinitiator, and was determined by 1H NMR measurement (Figure S3 and Table S1). All of these polymers showed monomodal GPC traces with relatively narrow molecular weight distributions (Figure S4). Thermally Induced Aggregation of the Copolymers. Like their PEO114-derived analogues, the block copolymers PEO45-b-PtNEA are water-soluble at low temperature and tend to aggregate upon heating above their CATs due to the dehydration of PtNEA block. Although the CATs of these block copolymers differ with PtNEA lengths, they are much lower than 37 °C, which has been proven by the CATs of the block copolymers PEO114-b-PtNEA or the cloud points of a series of PtNEA homopolymers with different chain lengths (Figure S5).57 C
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vesicular morphology. For copolymer P3 with the intermediate PtNEA length, clusters formed from the spherical nanoparticles were apparently observed. When quickly heating the copolymer solutions at a higher concentration (2.0 mg/mL), we observed different results. The concentrated aggregate dispersions were too cloudy to obtain reliable data by direct LLS measurements, except for P1, therefore, the concentrated dispersions of P2−P5 aggregates were diluted to 0.02 mg/mL at 37 °C and incubated for additional ∼2 h at this temperature prior to the measurements. As shown in Table 1 and Figure S8, at 2.0 mg/mL, P1 also formed spherical nanoparticles with a slight increase in hydrodynamic radius (or diameter) compared to that prepared at 0.2 mg/mL. However, the hydrodynamic radius (Rh2) of P2−P4 aggregates formed at 2.0 mg/mL was much larger than that at 0.02 mg/mL, which can be attributed to the association and/or fusion of the spherical nanoprticles or polymersomes as proven by the TEM images (Figure 1 and Figure S8). At 2.0 mg/mL, P5 formed polymersomes with a larger Rh2 compared to those prepared at 0.02 mg/mL. These polymersomes were stable with no obvious association or fusion. When the concentrated dispersion (2.0 mg/mL) was diluted to 0.2 mg/ mL at 37 °C before preparing the TEM sample, the isolated polymersomes were clearly observed (Figure 1d and Figure S9a). We also prepared P5 polymersomes at a polymer concentration of 0.05 mg/mL and found that their sizes (100− 160 nm) were greatly reduced as compared to the polymersomes (170−290 nm) formed at 2.0 mg/mL (Figure S9b). This agrees well with the results of other block copolymers.19,63 Finally, the vesicular structure of P5 polymersome was confirmed by using freeze-fracture TEM (Figure S10). Upon cooling, P5 polymersomes remained stable above 20 °C, below which they gradually disassembled, and finally dissociated completely into single polymer chains at ∼12 °C (Figure S12). By summarizing the above results, we conclude that the morphology of the copolymer aggregates gradually evolve from spherical nanoparticle to polymersome as the DP of the PtNEA block increased from 22 to 172, with the formation of rod-like structure at the intermediate PtNEA length. This trend is in accordance with the results of other thermosensitive block copolymers.29,59,64 Acid-Triggered Dissociation of the Copolymer Aggregates. The dissociation behaviors of P2 spherical nanoparticle and P5 polymersome were first studied by the light scattering method in aqueous solutions of different pHs at 37 °C. At pH 7.4, the scattered light intensity of the aggregate dispersions and Rh of the aggregates did not change significantly in the studied time period (5 h) for both P2 and P5 aggregates, indicating their stability upon incubation at the physiological pH (Figure 2 and S13a). However, the scattered light intensity of the copolymer dispersions greatly decreased under acidic conditions due to the dissociation of the aggregates, which is caused by the hydrolysis of ortho esters and the subsequent increase in hydrophilicity of the thermosensitive PtNEA block.57 For a specific copolymer, the dissociation rate of the aggregates greatly increased with decreasing pH of the medium. At the same pH, the P2 nanoparticle dissociated faster than the P5 polymersome, which was supported by their time-dependent 1H NMR spectra in the acidic deuterated buffer at 37 °C (Figure S14). Our results on a family of PtNEA homopolymers showed that the longer PtNEA had a larger phase transition enthalpy than the shorter one (data not shown). Therefore, this difference can be mainly
Regarding the double hydrophilic thermosensitive block copolymers, their thermally induced aggregation behaviors are influenced by various factors including heating rate, polymer concentration, block lengths, and so forth. Our previous data showed that, for the copolymers PEO114-b-PtNEA, a slow heating procedure resulted in the formation of large aggregates, and even macroscopic precipitation for the copolymer with a longer PtNEA block.57 Since a fast heating rate is generally helpful to obtain smaller particles, which are preferred for the systemic drug delivery, we adopted a fast heating protocol to prepare the aggregates of copolymers PEO45-b-PtNEA.19,21,58,59 In this protocol, the polymer solutions at ∼4 °C were quickly heated to 37 °C (∼5 min) and incubated for additional ∼2 h at this temperature prior to various measurements. In order to evaluate the aggregation ability of these block copolymers (P1P5) above the CATs, their critical aggregation concentrations (CACs) in aqueous solution at 37 °C were measured by the fluorescence method using pyrene as a probe (Figure S6 and Table 1). The values of CAC decreased from ∼0.1 mg/mL for P1 to ∼0.01 mg/mL for P2 with increasing DP of the PtNEA block up to 44, beyond which leveled off. The weak aggregation tendency of P1 is attributed to its shortest PtNEA block, which is consistent with the observation for other thermosensitive copolymers.58 Above their CATs in aqueous solutions, copolymers P1−P5 can self-assemble into various aggregates including spherical solid nanoparticle, cluster with rod-like structure and polymersome, which are mainly dependent on the PtNEA length and the polymer concentration. The morphologies and sizes of the copolymer aggregates were studied by LLS and TEM (Table 1, Figure 1, S7 and S8). At a low concentration (0.02 mg/mL for
Figure 1. TEM images of P2 (a) and P5 (b) at a polymer concentration of 0.2 mg/mL and P2 (c) and P5 (d) at a polymer concentration of 2.0 mg/mL.
P2−P5 and 0.2 mg/mL for P1), the hydrodynamic radii (Rh1) of the copolymer aggregates did not change much, being in the range of 99−115 nm. However, the Rg/Rh ratios of these aggregates significantly varied for these copolymers. For P1 and P2, the two copolymers with shorter PtNEA block, their Rg/Rh ratios are ∼0.70, demonstrating that their aggregates are spherical nanoparticles in morphology.60 For the copolymers with longer thermosensitive blocks (P4 and P5), the ratios are around 1.0, implying the formation of polymersomes.61,62 This speculation was further supported by their TEM images (Figure 1 and Figure S8). Spherical nanoparticles were observed for the aggregates of P1 and P2, while P4 or P5 showed a clear D
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drastic decrease in scattered intensity, and was supported by the results of NR fluorescence measurements (Figure 3). At pH 4.6, Rh of the P5 polymersomes deceased gradually, and the ratio of Rg/Rh increased up to ∼1.7 in 90 min, beyond which the light scattering data were too complicated to obtain reliable values of Rh and Rg (Figure S13d). At the end of hydrolysis, besides the hydrolyzed single chains (
