Fabrication of Functional Nano-objects through RAFT Dispersion

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Fabrication of Functional Nano-objects through RAFT Dispersion Polymerization and Influences of Morphology on Drug Delivery Liang Qiu, Chao-Ran Xu, Feng Zhong, Chun-Yan Hong,* and Cai-Yuan Pan* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui, People’s Republic of China S Supporting Information *

ABSTRACT: To study the influence of self-assembled morphologies on drug delivery, four different nano-objects, spheres, nanorods, nanowires, and vesicles having aldehdye-based polymer as core, were successfully prepared via alcoholic RAFT dispersion polymerization of p-(methacryloxyethoxy)benzaldehyde (MAEBA) using poly((N,N′-dimethylamino)ethyl methacrylate) (PDMAEMA) as a macro chain transfer agent (macro-CTA) for the first time. The morphologies and sizes of the four nano-objects were characterized by TEM and DLS, and the spheres with average diameter (D) of 70 nm, the nanorods with D of 19 nm and length of 140 nm, and the vesicles with D of 137 nm were used in the subsequent cellular internalization, in vitro release, and intracellular release of the drug. The anticancer drug doxorubicin (DOX) was conjugated onto the core polymers of nano-objects through condensation reaction between aldehyde groups of the PMAEBA with primary amine groups in the DOX. Because the aromatic imine is stable under neutral conditions, but is decomposed in a weakly acidic solution, in vitro release of the DOX from the DOX-loaded nano-objects was investigated in the different acidic solutions. All of the block copolymer nano-objects show very low cytotoxicity to HeLa cells up to the concentration of 1.2 mg/mL, but the DOX-loaded nano-objects reveal different cell viability and their IC50s increase as the following order: nanorods-DOX < vesicles-DOX < spheres-DOX. The IC50 of nanowires-DOX is the biggest among the four nano-objects owing to their too large size to be internalized. Endocytosis tests demonstrate that the internalization of vesicles-DOX by the HeLa cells is faster than that of the nanorods-DOX, and the spheres-DOX are the slowest to internalize among the studied nano-objects. Relatively more nanorods localized in the acidic organelles of the HeLa cells lead to faster intracellular release of the DOX, so the IC50 of nanorods is lower than that of the vesicles-DOX. KEYWORDS: endocytosis, aromatic imine, morphologies, p-(methacryloxyethoxy) benzaldehyde, doxorubicin, RAFT dispersion polymerization

1. INTRODUCTION Nanomedicine is a highly active and rapidly developing field due to a significant impact on diagnosis and therapeutics for treatment of human diseases.1−4 A number of drug delivery vehicles, such as polymeric micelles and organic/inorganic nanoparticles, have been developed for therapeutic treatments in oncology and cardiovascular diseases because such vehicles offer the potential to increase drug circulation time, improve drug solubility, prolong drug residence time in tumors, and reduce side effects.5−7 As one of drug delivery systems with the most potential, polymeric nano-objects have received great attention owing to their unique properties, such as high loading © 2016 American Chemical Society

capacity, increasing solubility of the drug in water, reduction of systemic adverse effects, and promotion of their accumulation at the tumor sites via the enhanced permeability and retention effect.8−12 The parameters for optimizing the in vivo performance of these vehicles include the choice of polymer, the size and morphology of the nanoparticles, and the surface chemistry, which govern functional behaviors of the vehicles.13−15 Because the size of nanoparticles strongly Received: April 20, 2016 Accepted: June 29, 2016 Published: July 11, 2016 18347

DOI: 10.1021/acsami.6b04693 ACS Appl. Mater. Interfaces 2016, 8, 18347−18359

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ACS Applied Materials & Interfaces

selected poly(p-(methacryloxyethoxy)benzaldehyde) (PMAEBA) as the core polymer of the nanoparticles because aminecontaining drug can be linked to the cores of nanocarriers via an acid-labile aromatic imine linkage. In addition, reaction between aldehyde of the PMAEBA and diamine compounds produced cross-linked nanoparticles, resulting in superior stability of the morphologies even when they are subject to high dilution, media changes, and large shear forces upon intravenous injection. Different from the traditional self-assembly method, the polymerization-induced self-assembling (PISA) produces various morphologies directly from the polymerization; thus, a large quantity of the expected morphologies can be obtained from one-pot polymerization.47−57 The most studied polymerization for PISA is reversible addition−fragmentation transfer dispersion polymerization (RAFTDP), and fabrication of the pure morphologies including spherical micelles, nanorods or nanowires, and vesicles can be achieved by changing the feed molar ratio, monomer concentration, and solvent.52−55 Therefore, in this work, we studied fabrication of the pure spheres, nanorods, nanowires, and vesicles through RAFTDP of the MAEBA in ethanol using poly((dimethylamino)ethyl methacrylate) (PDMAEMA) as macro chain transfer agent (macroCTA), subsequently covalently attached doxorubicin (DOX) to the core polymer of the nano-objects by reaction of aldehyde groups in the cores with the primary amine of DOX, and then studied the in vitro DOX release behaviors, DOX distribution, and cytotoxicity in HeLa cells

correlates with their biodistribution, tissue penetration, and cell uptake, the size impacts antitumor efficacies.16,17 For example, study on the poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGlu) spherical micelles with diameters in the range of 30−100 nm showed that only the 30 nm spheres penetrated poorly permeable pancreatic tumors to achieve an antitumor effect although all the polymer micelles penetrated highly permeable tumors in mice.17 Surface chemistry mainly affects the interactions of particles with the cells and the tissues in the body, and one strategy widely used in the surface chemistry is surface modification: for example, targeting carriers to the specific tissues has been extensively used.18,19 However, most of the nanoparticles applied to drug delivery are spheres; influences of the nanoparticle morphology on drug delivery have received little attention although some reports indicate that not only size but also the morphology of the nanoparticles has significant influence on the efficiency of drug delivery.12,15,20−24 Discher and co-workers prepared filomicelles and spheres from diblock copolymers, PEG-b-polyethylethylene or PEG-b-polycaprolactone, and observed that the filomicelles persisted in the circulation up to 1 week after intravenous injection, which is about 10 times longer than their spherical counterparts.20 Liu et al. prepared four different morphologies: spheres, smooth discs, large compound vesicles (LCVs), and staggered lamellae, from the PEG-b-poly(camptothecin-based methacrylate), and the staggered lamellae revealed extended blood circulation duration, the fastest cellular uptake, and unique internalization pathways in comparison with the other three nanostructure types.21 Boyer et al. studied the influence of nanoparticle shapes on drug delivery; the results demonstrate that the drug delivery performance of polymeric nanorods is superior to that of the nanospheres,23 and the gold nanorods and nanostars functionalized with doxorubicin (DOX)-conjugated polymer exhibited higher cytotoxicity in comparison with the same polymer-functionalized gold nanosphers.24 Thus, studying the influence of the nanoparticle morphology on drug delivery is of particular interest. As a key parameter, the polymers, especially a variety of biodegradable and stimuli-responsive polymers, have been extensively investigated as drug vehicles.25−28 Generally, two strategies are used to load the hydrophobic drug into the polymeric nanoparticles. One is encapsulation of the drug into the hydrophobic core of the nanoparticles for improvement of the water solubility and enhancement of the drug stability.25,29−32 The other is to covalently conjugate the drug to the core of the nanoparticles via a labile linkage, such as photocleavable linker, reducible linker, and so on, that can be cleaved under environmental stimuli.28,33−35 Among the stimuli-responsive nanocarriers, the pH-responsive polymeric vehicles have received extensive interest owing to their particular relevance in biological applications.31,32 The acidcleavable bonds used for covalently linking the drug onto the nanoparticles include acetal,36−38 hydrozones,39−41 and imines because the pH value of the tumor cells is lower than the normal cells.42 Compared to synthesis of other acid-sensitive linkages, the imine bond is easier and more efficient to synthesize43 although this linkage is scarcely utilized to link the drug onto the polymeric nanocarriers due to cleavage at physiological pH.44 Recent studies indicate that aromatic imines with extended π−π conjugation are quite stable at physiological pH, while they are easily hydrolyzed in weakly acidic solutions.45,46 Therefore, in this study, in order to study the influence of the polymer morphologies on drug delivery, we

2. EXPERIMENTAL SECTION 2.1. Materials. Doxorubicin hydrochloride (DOX·HCl, Aladdin) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma) were purchased and used as received. p(Methacryloxyethoxy)benzaldehyde (MAEBA) was synthesized according to the method reported, and its 1H NMR data are consistent with the results reported elsewhere.58 4-Cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to the method reported in our previous work.27 Azobis(isobutyronitrile) (AIBN, Aldrich) was recrystallized from ethanol. 2-((N, N-Dimethylamino)ethyl)methacrylate (DMAEMA, 97%, Alfa) and tetrahydrofuran (THF) were purified according to the method in our previous report.27 All other solvents with analytical grade were purchased from Shanghai Chemical Reagent Co. and used without purification. 2.2. Characterization. 1H NMR spectra were acquired on a Bruker 400 MHz spectrometer in either CDCl3 or DMSO-d6. UV/vis spectra were measured on a Unico UV/vis 2802PCS spectrophotometer (United Products & Instruments, Inc., Dayton, NJ, USA). Transmission electron microscopy (TEM) images were obtained on JEM-100SX TEM operating at 100 kV. The samples for TEM observation were prepared by placing a dilute copolymer solution (5.0 μL) on a carbon-coated copper grid and drying at room temperature. Scanning electron microscope (SEM) images were acquired on a JEOL JSM-6700F. The samples for SEM measurements were prepared by placing a drop of the nanoparticle solution in ethanol on copper grids and then gilding a shell of Pt nanoparticles. Dynamic light scattering (DLS) measurements were performed on a DynaPro light scattering instrument (DynaPro-99E) at 25 °C with 824.3 nm laser. Molecular weight and Mw/Mn were determined on a Waters 150C gel permeation chromatography (GPC) equipped with microstyragel columns and RI 2414 detector at 30 °C, monodispersed polystyrene standards were used in the calibration of Mn, Mw, and Mw/Mn, and DMF was used as eluent at a flow rate of 1.0 mL/min. 2.3. Preparation of PDMAEMA Macro-CTAs. A general procedure for synthesis of macro-CTA, PDMAEMA is as follows. A solution containing DMAEMA (4 g, 25.5 mmol), CPADB (71 mg, 0.255 mmol), AIBN (6 mg, 0.04 mmol), and THF (4 mL) was added 18348


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Scheme 1. Chain Extension of a PDMAEMA Macro-CTA with MAEBA by RAFTDP at 70 °C To Produce Stabilized PDMAEMA−PMAEBA Diblock Copolymer Nano-objects through Polymerization-Induced Self-assembly (A) and Illustration of the Prodrug Nanoparticles and Their pH-Responsive Intracellular Release (B)

filtration. After drying in a vacuum oven at room temperature overnight, the polymer was obtained. For TEM and DLS measurements, a small portion of the reaction mixture was diluted with ethanol, and the TEM sample was prepared by depositing a drop of the diluted dispersion onto a copper grid, subsequently drying at room temperature. The remaining mixture was used in investigation of drug delivery and cytotoxicity. 2.5. Preparation of Various Prodrug Nano-objects. DOXconjugated nano-objects via acid-labile aromatic imine were prepared by condensation reaction of aldehyde in the core MAEBA units of nano-objects with amine of the DOX. In a typical example, various polymeric nano-object dispersions obtained were diluted with ethanol of one-third dispersion volume. Into a two-necked flask, triethylamine (50 mol % MAEBA in the polymer), polymeric nano-object dispersion (10 mL), and DOX·HCl (50 mol % MAEBA in the polymer) were added; the condensation reaction was carried out at room temperature for 6 h while stirring. Subsequently, the reaction mixture was dialyzed (Mw cutoff, 3500 Da) against ethanol; after dialysis for 24 h, the mixture was dialyzed against buffer solution (pH = 7.4) for another 24 h. The drug-loading content (DLC) was measured using UV/vis quantitative method, and the standard curve was acquired by plotting the UV absorbance at 497 nm against different DOX concentrations in deionized water. 2.6. In Vitro Release of DOX. The release profile of DOX from the DOX-loaded nano-objects was conducted under different pHs (pH = 5, or 6, or 7.4) at room temperature. In a typical experiment, 2 mL of

to a glass vessel equipped with a magnetic stirring bar. After the tube was degassed through three freeze−pump−thaw cycles and then sealed, the polymerization tube was placed in a preheated oil bath at 70 °C for 8 h. The PDMAEMA was isolated by precipitation in nhexane followed by filtration and then dried under vacuum overnight. The final conversion (45%) was determined by 1H NMR spectroscopy. 2.4. Linear Macro-CTA Mediated RAFT Dispersion Polymerization. A series of RAFTDPs with feed molar ratios of MAEBA/ PDMAEBA45 ranging from 50 to 250 and PDMAEMA45/AIBN = 1/ 0.2 at 10 wt %, or 15 wt %, or 20 wt % MAEBA content in ethanol were carried out at 70 °C for 18 h. A typical procedure is described as follows. PDMAEMA45 (28 mg, 4 μmol), AIBN (0.132 mg, 0.8 μmol), and MAEBA (234 mg, 1 mmol) were dissolved in ethanol (1.049 g), and then the solution was added into a 5 mL reaction vessel. After the vessel content was degassed through three freeze−pump−thaw cycles, the vessel was sealed and then placed in a preheated oil bath at 70 °C for 18 h. The polymerization was stopped by rapidly cooling the vessel to room temperature. A drop of the reaction mixture was taken for 1H NMR measurement, and the MAEBA conversion was calculated based on the integral values of the aldehyde proton signal at 9.56 ppm and the ester methylene proton signal at 3.8−4.12 ppm. To measure 1H NMR spectra and GPC curves of the diblock copolymers, PDMAEMA45-b-PMAEBA, a small amount of the reaction mixture was diluted with THF, and the diluted solution was dropped into excess n-hexane while stirring; then the precipitate was obtained by 18349


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Figure 1. 1H NMR spectrum (A) and GPC trace (B) of the PDMAEMA45 prepared by RAFT polymerization of the DMAEMA with a feed molar ratio of DMAEMA/CPADB/AIBN = 100/1/0.11 in THF at 70 °C for 8 h.

Table 1. Results Obtained from RAFTDP of MAEBA Using PDMAEMA45 as Macro-CTAa DLSe

GPC solid contents (%) 10 10 10 10 10

b

target composition

conv (%)

PDMA45-PMAEBA50 PDMA45-PMAEBA100 PDMA45-PMAEBA150 PDMA45-PMAEBA200 PDMA45-PMAEBA250

100 100 100 100 99.2

c

DPPMAEBA

d

50 100 152 201 248

Mn (kg/mol)

Mw/Mn

Dh (nm)

PDI

morpf

25400 35800 51500 64200 76200

1.49 1.43 1.44 1.52 1.55

57 − − − 137

0.241 − − − 0.184

S S+N N N+L V

The polymerization with feed molar ratio of PDMAEMA/AIBN = 1/0.2 in ethanol at 70 °C for 18 h. bCalculated based on the weights of MAEBA and ethanol. cConv is an abbreviation of conversion, which was measured by 1H NMR data. dDPPMAEBA is an abbreviation of degree of polymerization of PMAEBA, which was calculated based on 1H NMR data. eDh and PDI respectively refer to diameter and polydispersity index of the nano-objects, which were determined by dynamic light scattering; “−” is used because the real sizes of nanowires cannot be obtained by DLS method. fMorp refers to morphology: S, sphere; N, nanowire; L, lamella; V, vesicle. a

(4,5-dimethyliazolyl-2)-2,5-diphenyltetrazolium bromide] (MTT) assay. The cells were seeded in 96-well plates at a density of 5000 cells per well, and after being incubated for 24 h, the cells were treated with various concentrations of DOX·HCl, the nano-objects, and the DOX-conjugated nano-objects in 96-well plates. The culture medium in each well was removed and replaced by 100 μL of DMSO. The plate was gently agitated for 15 min, and the absorbance values were recorded at a wavelength of 490 nm on a Thermo Electron MK3 instrument. The cell viability was calculated as A490,treated/A490,control × 100%, where A490,treated and A490,control are the absorbance values with or without addition of the nano-objects or DOX-nano-objects, respectively. Each experiment was done in triplicate. The data were recorded as the mean value plus a standard deviation (±SD).

DOX-loaded nano-object dispersion (2 mg/mL) was add into a dialysis bag (Mw cutoff, 3500 Da) and then immersed in 60 mL of PBS solution at pH = 5.0, or 6.0, or 7.4 in a beaker. The beaker was kept at 25 °C with constant stirring (300 rpm). At predetermined time intervals, 2 mL of the PBS dialysis solution was taken out for estimation of the released DOX, while 2 mL of the fresh PBS solution was added into the beaker. UV absorbance at 497 nm of the solutions obtained at different dialysis time was measured and the DOX releasing profile was obtained. A series of parallel experiments were carried out. Each measurement was done in triplicate, and the data were recorded as the mean value plus a standard deviation (±SD). 2.7. Intracellular DOX Release. Intracellular DOX release from the DOX-loaded nano-objects was tested using a confocal laser scanning microscopy (CLSM). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 96-well plate under an atmosphere with 5% CO2 at 37 °C for 24 h and then changed to the freshly prepared DMEM with the concentration of the DOX-loaded nanosphere solution (2 mg/mL), which is equivalent to 0.23 μg/mL DOX concentration. After being treated for 1 h, or 2 h, or 3 or 4 h, the culture medium was removed, after being rinsed with PBS two times; the cells were fixed with formaldehyde. After the cells were stained with the DAPI (blue) or with the Lyso Tracker green DND (green) and then rinsed with PBS buffer; the cell images were acquired on a CLSM (Leica TCP SP5) at 595 nm (Ex = 485 nm). For the tests of other DOX-loaded nano-objects and free DOX, the same procedure and the same concentration of DOX in the nano-objects were used. When the HeLa cells were treated with free DOX and three DOXloaded nano-objects for 24 h, all of the nano-object concentrations used are equivalent to 0.1 μg/mL DOX concentration in order to reduce the cell death. 2.8. Cytotoxicity Assay. HeLa cell was chosen for the tests. All the cells were first cultured in DMEM supplemented with 10% FBS under an atmosphere with 5% CO2 at 37 °C. The DOX·HCl, various nano-object dispersions and the corresponding DOX-loaded nanoobject dispersions were tested using the standard thiazolyl blue [3-

3. RESULTS AND DISCUSSION 3.1. Fabrication of PDMAEMA-b-PMAEBA Diblock Copolymer Nano-objects. Similar to the synthetic strategy for preparation of various nano-objects through the RAFTDP,55,59,60 the synthetic strategy in this study includes two steps of RAFT polymerization, the preparation of PDMAEMA macro-RAFT agent by RAFT polymerization of DMAEMA and fabrication of various diblock copolymer nanoobjects by RAFTDP of MAEBA using PDMAEMA as macroRAFT agent, as shown in Scheme 1A. The PDMAEMA macro-RAFT agent was prepared by RAFT polymerization of DMAEMA with a feed molar ratio of DMAEMA/CPADB/AIBN = 100/1/0.12 in THF at 70 °C for 8 h, and the structure of the resultant polymer is verified by its 1 H NMR spectrum, as shown in Figure 1A; the proton signals at δ = 7.86, 7.50, and 7.36 ppm (a) and δ = 4.03 ppm (b) are respectively ascribed to the aromatic protons of dithiobenzoate and the ester methylene protons of DMAEMA units, ascription for other proton signals of the PDMAEMA are marked in this 18350


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Figure 2. 1H NMR spectra (A′) and GPC traces (B′) of the block copolymers: (A) PDMAEMA45-b-PMAEBA50, (B) PDMAEMA45-b-PMAEBA100, (C) PDMAEMA45-b-PMAEBA150, (D) PDMAEMA45-b-PMAEBA200, and (E) PDMAEMA45-b-PMAEBA250 prepared by RAFTDP of the MAEBA (PDMAEMA/AIBN = 5/1, molar ratio) at 10 wt % monomer content in ethanol at 70 °C for 18 h.

Figure 3. Representative TEM images of the PDMAEMA45-b-PMAEBAn [n = 50 (A), n = 100 (B), n = 150 (C), n = 200 (D), and n = 250 (E)] nano-objects prepared by RAFTDP at a total solid concentration of 10 wt % in ethanol at 70 °C for 18 h.

figure. The number-average molecular weight (Mn,NMR) was calculated based on the integration ratio of the proton signals at 4.03 (b) and 7.36−7.86 ppm (a) and is 7000 g/mol; its degree of polymerization (DP) is approximately 45. Its DMF GPC curve in Figure 1B reveals Mn = 8200 g/mol and Mw/Mn = 1.32. Based on the molar ratio of DMAEMA/CPADB and conversion, the theoretical DP is 45, and the resultant PDMAEMA45 was used as macro-CTA in the subsequent RAFTDP because of its high hydrophilicity. With the same procedure, PDMAEMA95 was prepared and its characterization results are shown in Figure S1. As mentioned in the Introduction, the aldehyde group of PMAEBA can be conveniently used in the modified reactions based on the requirement of applications, which is the reason for use of MAEBA as the core-forming monomer. In addition,

MAEBA is soluble, but its polymer is insoluble in ethanol, which is requisite for dispersion polymerization. So, preparation of different nano-objects through RAFTDP of the MAEBA is feasible. Based on our previous study,61 the monomer conversion in the RAFTDP was dependent on the feed molar ratio of monomer/macro-CTA, almost complete monomer conversion, and pure spherical micelles, nanorods, and vesicles of the nano-objects can be obtained at lower feed ratios. Thus, the feed molar ratios of MAEBA/PDMAEBA45 in the range of 50−250 were used in the RAFTDP at 70 °C for 18 h, and the results are listed in Table 1 and Table S1 in Supporting Information. We can see that all of the monomers almost completely converted after 18 h (96−100%), which is consistent with the reported result that all-methacrylic formulations had very high monomer conversions.62 18351


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Figure 4. TEM images (A−H) and SEM images (A*−H*) of the PDMAEMA45-b-PMAEBA50-15 spheres (A and A*) and the DOX-loaded spheres (E and E*), PDMAEMA45-b-PMAEBA50-20 nanorods (B and B*) and the DOX-loaded nanorods (F and F*), PDMAEMA45-b-PMAEBA100-20 nanowires (C and C*) and the DOX-loaded nanowires (G or G*), and PDMAEMA45-b-PMAEBA250-10 vesicles (D and D*) and the DOX-loaded vesicles (H and H*).

The same with the RAFTDP reported,47−55 a series of diblock copolymers, PDMAEMA45-b-PMAEBAn (n = 50, 100, 150, 200, and 250) were produced by chain-extending PDMAEMA45 macro-CTA with MAEBA via RAFTDP with various feed molar ratios at different total solid contents in ethanol at 70 °C for 18 h, and their 1H NMR spectra are shown in Figure 2A′. All proton NMR spectra in Figure 2A′ show characteristic proton signals of PMAEBA at δ = 9.89 (a), 7.87 (b), 7.05 (c), and 4.29 ppm (d), which are respectively attributed to the aldehyde proton, the aromatic protons, and the ester methylene proton of the MAEBA units, and the characteristic proton signals of the PDMAEMA appear respectively at δ = 4.06 (g), 2.57 (e), and 2.27 ppm (f), which correspond to the ester methylene protons, the methylene and methyl protons next to the nitrogen, respectively, and the other proton signals are marked in this figure. Thus, the diblock copolymers have been successfully formed. Their Mn,NMRs and the DPs of PMAEBAs (DPPMAEBAs) were calculated based on the integral values of the signals at δ = 9.89 (a) and 2.57 ppm (e), and the Mn,NMR of PDMAEMA or the DPPDMAEMA; the results listed in Table 1 and Table S1 demonstrate that all of the DPPMAEBAs are close to their corresponding feed molar ratio of MAEBA/PDMAEMA owing to almost complete MAEBA conversions. GPC traces of the block copolymers, PDMAEMA45-bPMAENAn (n = 50, 100, 150, 200, and 250) in Figure 2B′ and Figure S2 are invariably unimodal with little or no tailing,

which indicates relatively high blocking efficiency, and no significant chain radical termination throughout the polymerization. However, their molecular weight distributions are relatively broad (Mw/Mn = 1.43−1.60); the exact reason is unknown. In the RAFTDP, various copolymer nano-objects are generated via in situ self-assembly by varying DP of the core polymers. The spherical micelles, the nanorods or nanowires, and the vesicles are formed at different reaction times owing to DP increase of the core polymer chains with the polymerization time.55 Because DPPMAEBA is dependent on the feed molar ratio of MAEBA/PDMAEMA, various copolymer nano-objects could be produced by varying the feed molar ratio of MAEBA/PDMAEMA. Figure 3 is the TEM images of various nano-objects constructed of PDMAEMA45-b-PMAEBAn obtained by RAFTDP with the feed molar ratios (50, 100, 150, 200, and 250) of MAEBA/PDMAEMA45 at the total solid content of 10 wt % in ethanol. The block copolymer with DPPMAEBA = 50 forms spherical micelles as shown in Figure 3A; when the DPPMAEBA increases to 100, nanowires begin to appear (Figure 3B). At DPPMAEBA = 152, pure nanowires are observed (Figure 3C). At DPPMAEBA = 201, we see an interesting morphology, bilayer octopi with radial “tentacles”, which are formed by partial coalescence of highly branched nanowires (Figure 3D). This is one intermediate of the nanowires-to-vesicles transition, which was observed in the previous report.54 When the chain length of PMAEBA increases 18352


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ACS Applied Materials & Interfaces Table 2. Characterizations of the DOX-Loaded Nano-objects polymera

morphologyb

DDLS (nm)c

PDIc

DTEMd (nm)

DLC (%)e

PDMA45-PBA50-15 DOX-PDMA45-PBA50-15 PDMA45-PBA50-20 DOX-PDMA45-PBA50-20 PDMA45-PBA100-20 DOX-PDMA45-PBA100-20 PDMA45-PBA250-10 DOX-PDMA45-PBA250-10

spheres spheres nanorods nanorods nanowires nanowires vesicles vesicles

70 77 − − − − 137 176

0.212 0.324 − − − − 0.184 0.228

63 70 19/140 20/160 20/∼2000 20/∼2000 100 180

0 11.5 0 13.9 0 14.7 0 16.2

eff (%)f 19.3 23.3 22.7 23.7

a

All PDMA45-PBAn-10 (-15 or -20) means PDMAEMA45-b-PMAEBAn, the numbers (10, or 15, or 20) after the last short line refer solid contents (wt %), and all PDMA45−PBAn nano-objects were prepared by RAFTDP of MAEBA (PDMAEMA/AIBN = 5/1, molar ratio) in ethanol at 70 °C for 18 h. bThe morphology observed by the TEM and SEM. cDDLS and PDI respectively refer to average diameters (D) and size distribution of the PDMA45−PBAn or the DOX-loaded PDMA45-PBAn nano-objects in water, which were measured by DLS method. dDTEM was measured by TEM images of the nano-objects. eDLC refers to drug-loaded content, which was determined by ultraviolet quantitative analysis. fEff means reaction efficiency, which was calculated according to the following equation: Eff (%) = (measured DLC)/(theoretical DLC).

Figure 5. 1H NMR spectra of the PDMAEMA45-b-PMAEBA250 (A) and PDMAEMA45-b-PMAEBA250-DOX (B) in DMSO-d6.

PMAEBA50 in RAFTDP at a total solid content of 20 wt % produced nanorods containing spheres and a few longer nanowires (Figure S4A), After removing the spheres by centrifugation, then the longer nanowires were removed by centrifugation at 10000 rpm for 15 min in order to avoid an influence of the nanowires on the drug delivery. TEM and SEM images of the resultant nanorods are respectively shown in Figure 4B,B*; their sizes were measured, and the results are listed in Table 2. The vesicles fabricated from PMAEMA45-bPMAEBA250-10 reveal a relatively small size (D = 137 nm) in all the vesicles obtained (see Table 1 and Table S1). For understanding the influence of the nano-objects’ size on cell internalization, nanowires with D = 20 nm and length ∼ 2000 nm were used for comparison. The morphologies and the sizes of all these samples are respectively supported by their TEM and SEM images as shown in Figure 4. The TEM (Figure 4A) and SEM (Figure 4A*) images reveal that the in situ selfassembling of PMAEMA45-b-PMAEBA50-15 forms spheres with approximately 63 nm. The D and length of PDMAEMA45-bPMAEBA50-20 nanorods are 19 and 140 nm, respectively, and the D of PDMAEMA45-b-PMAEBA250-10 vesicles is 137 nm. So, the sizes of all of the nano-objects used in the drug delivery studies are less than 200 nm besides the nanowires. The condensation reaction of primary amine with aldehyde under mild condition is well-known in organic chemistry and was used to conjugate the anticancer drug DOX onto the cores of nano-objects via acid-labile imine linkage as shown in Scheme 1A. To identify whether this reaction occurs, 1H NMR spectra of the PDMAEMA45-b-PMAEBA200-DOX and its precursor are respectively shown in panels B and A of Figure 5. Besides the characteristic proton signals of the DMAEMA

continuously to DPPMAEBA = 248, vesicles are formed as shown in Figure 3E. Figures S3 and S4 are representative TEM images of the PDMAEMA45-b-PMAEBAn nano-objects respectively prepared from the polymerizations at total solid contents of 15 and 20 wt %; A similar sphere-to-nanowire-to-vesicle transition is observed with an increase of DPPMAEBA. When the polymerizations are conducted with the same feed molar ratio of MAEBA/PDMAEMA45, with increasing solid contents, the transitions occur at relatively low targeting DPPMAEBA. To study the influence of copolymer morphology on drug delivery and further to better design drug carriers, the first step is fabrication of pure nano-objects. In this study, the pure spheres, nanorods, and vesicles were selected because the three morphologies have been extensively studied, but their difference in drug delivery has never been investigated systematically. 3.2. Conjugation of DOX onto Various Nano-objects. Because the endocytosis tests revealed an upper size limit of approximately 200 nm for internalization,63 we tried to select the spheres, nanorods, and vesicles with average diameter (D) of less than 200 nm from the obtained nano-objects for studying the influence of the morphologies on drug delivery. Although DLS is extensively used to measure the sizes of the nano-objects, the DLS sizes of the nanorods and nanowires are not real sizes, so the sizes measured by TEM are also referenced in the selection of the nanorods. Although RAFTDP with feed molar ratio of MAEBA/PMAEMA = 50 at monomer contents of 10 and 15 wt % produced the spherical micelles (Table 1 and Table S1), PMAEMA45-b-PMAEBA50-15 (“-15” refers to the solid content) spheres were selected because their size (D = 70 nm) is more close to the other two morphologies (nanorods and vesicles). The in situ self-assembling of PDMAEMA45-b18353


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the nanorods (12.7%), the size increase of the vesicles (28.5%) is bigger. 3.3. In Vitro Release of DOX. As mentioned previously in the Introduction, the aromatic imine linkage is acid-labile, so the DOX in the DOX-loaded nano-objects can be triggered to release under the acidic environment. All the DOX-loaded nano-objects used in the drug release study were dialyzed against ethanol for 24 h and then neutral deionic water (pH = 7.4) for another 24 h in order to completely remove the unreacted DOX. The in vitro release tests of the four DOXloaded nano-objects were carried out in aqueous buffer solutions at pH = 7.4, 6.0, and 5.0, and the results are shown in Figure 6. When the drug release was performed at pH = 7.4,

and MAEBA units, we can see the appearance of the aromatic proton signals of DOX at δ = 8.03−7.6 ppm (k) and the methine proton signal of CHN bond at δ = 8.18−8.28 ppm (j), indicating that DOX is covalently linked onto the PMAEBA chains via imine linkage. In addition to the proton NMR method, the ultraviolet quantitative method was applied to measure their DLCs, which were estimated based on the absorbance change at 497 nm (Figure S7), and the results are listed in Table 2. Reaction efficiency of the aldehyde groups in the MAEBA units with DOX was calculated according to weight ratio of the DLC measured to the theoretical DLC; the latter was estimated assuming that the aldehyde groups of PMAEBA were completely reacted with DOX, and the results are listed in Table 2. Theoretically, this drug-loading strategy can produce a prodrug with ultrahigh DLC; for example, the theoretical DLC is 59.6% for PDMAEMA45-PMAEBA50-DOX and 68.2% for PDMAEMA45-PMAEBA250-DOX. However, the purpose of this study is to compare the drug delivery behaviors of the four nano-objects; a significant difference of their DLCs is not expected. So, all the condensation reactions were conducted at the same recipe and the same conditions; DLCs of the obtained DOX-loaded nano-objects increase from 11.5% to 16.2% with an increase of DPPMAEBA from 50 to 250 (Table 2), so the DLC difference among the four DOX-loaded nano-objects is small. Similarly, the reaction efficiencies, which are between 19.3% and 23.7% (Table 2), have little difference also, demonstrating that their morphologies do not significantly influence the reactivity of aldehyde. Conjugation DOX onto the core alters the structure of the PMAEBA chains, possibly leading to alternation of the morphologies and size of the nano-objects. Therefore, it is necessary to check whether the condensation reaction induces any change of the four nano-objects; TEM, SEM, and DLS were used for this purpose, and the results are shown in Figures 4 and S8. Panels E−H and E*−H* of Figure 4 are TEM and SEM images of the four DOX-loaded nano-objects, respectively. Compared to the images of their corresponding nanoobjects before conjugation reaction (panels A−D and A*−D* of Figure 4), all the morphologies after the reaction remain well; such as, the images in Figure 4F and 4F* are nanorods, which is consistent with their morphology before the reaction (Figure 4B,B*). Generally, the SEM image of vesicles looks like the spherical micelles; in order to verify the vesicles structure, the sample was crushed as we did in our previous study.64 As shown in Figure 4D*,H*, we can clearly see the vesicles structure from the crushed nano-objects. Although the morphologies do not change after drug loading, their sizes become bigger generally in comparison with their precursor; for example, panels A and E of Figure 4 reveal that the Ds of the spheres (PDMAEMA45-b-PMAEBA50-15) and their DOXloaded spheres are 63 and 70 nm, respectively. The DLS data and curves of the three nano-objects are listed in Table 2 and are shown in Figure S8, respectively. As we mentioned earlier, the DLS size of the nanorods is not the real size; their D value is not listed in Table 2, but the DLS curves are shown in Figure S8 just for studying the influence of the DOX loading on their sizes. All the results reveal the size increase of the nano-objects after conjugation reaction, which is the same with that obtained from TEM measurements, and the Ds of the spheres, nanorods, and vesicles after DOX loading increase respectively from 70, 150, and 137 nm to 77, 169, and 176 nm. Relative to the size increase of the spheres (10%) and

Figure 6. DOX release profiles of the vesicles-DOX (a), nanorodsDOX (b), nanowires-DOX (c), and spheres-DOX (d) in the aqueous buffer solutions at pH = 7.4 (a3, b3, c3, and d3), at pH = 6.0 (a2, b2, c2, and d2) and at pH = 5.0 (a1, b1, c1, and d1).

less than 1% of the DOX in the nano-objects was released, indicating that the aromatic imine bond is very stable under neutral condition, which is consistent with the reported result.45,46 This interesting property is expected for effectively avoiding premature drug release in blood circulation. However, when the pH of aqueous solution decreases to 6.0, the release of DOX becomes rapid, and much rapid release of the DOX from the nano-objects occurs at pH = 5.0 (Figure 6) owing to fast cleavage of the aromatic imine linkages in more acidic solution. The drug release profiles in Figure 6 reveal that the four nano-objects tested display almost the same drug release behavior: the fast drug release within the initial 10 h is observed, and then the drug release becomes slower. The four nano-object prodrugs show different drug release rates in the two acidic aqueous solutions, and their release rates at the same pH display as the following order: vesicles > nanorods ∼ nanowires > spheres. But the difference of their release rates is small; for example, the release rates within the initial 10 h and after 12 h at pH = 5.0 are respectively 22.0 and 1.14 μg/h (vesicles), 17.8 and 1.00 μg/h (nanorods), 17.1 and 1.01 μg/h (nanowires), and 12.8 and 0.95 μg/h (spheres). The slight difference of release rate between the different morphologies may be ascribed to a slight increase of the DLC from spheresDOX (11.5%) to nanorods-DOX (13.9%) and nanowires-DOX (14.7%) to vesicles-DOX (16.2%). 3.4. In Vtiro Cytotoxicity Assay. Cytotoxicity of the four different nano-objects or their corresponding DOX-loaded nano-objects was respectively evaluated by MTT assay, and HeLa cells were used in this study. The cells were incubated 18354


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with the IC50 values of 3.94 μg/mL for nanorods-DOX, 5.98 μg/mL for vesicles-DOX, and 11.42 μg/mL for DOX-spheres as shown in Figure 8. Because the three nano-objects have no obviuos cytotoxicity as shown in Figure 7, it is reasonable to deduce that cytotoxicity of the prodrug comes from release of the DOX in the nano-objects. Considering the entrance ability of the DOX released from the three different nano-objects into the nuclei is the same, their IC50 difference might be due to differences of the cellular internalization and the drug release in the cells. In order to clearly understand the IC50 difference, further studies are required. 3.5. Intracellular DOX Release. In order to investigate the cellular internalization of the nano-object prodrugs and the pHresponsive intracellular release of DOX from the DOX-loaded nano-objects, the HeLa cells were incubated with the solutions of three DOX-loaded nano-objects including spheres, nanorods, and vesicles at 37 °C for 24 h, and all the nano-object solutions had the same concentration of DOX, the incubated HeLa cells were observed by confocal laser scanning microscope (CLSM), and the results are shown in Figure 9. It was reported that the free DOX entered into the cells through passive diffusion without requirement for a specific transporter;11 due to low solubility of the DOX in an aqueous solution, entrance of the free DOX into the cells is slow, so the cells display very weak red as shown in the CLSM image of Figure 9A2. Because the DAPI is a cell-permeable DNA-binding dye, which can be used to microscopically detect the nuclei of cells, as shown in Figure 9A1, the nuclei of HeLa cells stained with DAPI exhibit strong blue fluorescence, and we can see relatively weak red fluorescence in the cell nuclei, indicating entrance of the DOX into the nuclei. For nanocarriers including the polymeric self-assemblies, endocytic pathways are the main mechanism for cellular internalization after binding to the cell surface.11 Internalization tests of the three DOX-loaded nano-objects were conducted by respectively treating the HeLa cells with the solutions of spheres-DOX, nanorods-DOX, and vesicles-DOX, and then respectively staining with the DAPI and Lyso Tracker green DND; their CLSM images are shown in Figure 9. Compared to Figure 9A2, red fluorescence of the HeLa cells incubated with the three DOX-loaded nano-objects is much stronger, demonstrating that more DOX-loaded nano-objects enter into the cells than the free DOX. Panels B2−D2 of Figure 9 reveal that the three DOX-loaded nano-objects are mainly localized in the cytoplasm of the HeLa cells, and by analysis of these CLSM images, we can observe an interesting phenomenon, the red fluorescent strength of the cells respectively treated with the spheres-DOX, nanorods-DOX, and vesiclesDOX has the following order: vesicles > nanorods > spheres. To further verify this phenomenon, the kinetics of cellular uptake was evaluated by quantification from CLSM analysis from 1 to 4 h incubation (Figure 10A), and the results are shown in Figure 10B. The uptake rates of three different nanoobjects are quite different, but their uptake rate order is the same with the result obtained from Figure 9B2−D2: the vesicles-DOX exhibit the fastest internalization rate, and the internalization of nanorods-DOX is slower than the vesiclesDOX but is faster than the spheres-DOX. However, the in vitro cytotoxicity tests revealed that the DOX-nanorod is more cytotoxic than the vesicles-DOX, indicating that, in the cells, the DOX molecules released from the nanorods-DOX are faster than that from the vesicles-DOX. As we mentioned before in the Introduction, the acidic environment is the prerequisite for

respectively with various concentrations of the four nanoobjects solutions at 37 °C for 24 h, and cells treated without the nano-objects were used as a control. The relationship of cell viability with the concentrations of the four different nanoobjects shown in Figure 7 displays virtually no toxicity of the

Figure 7. Relationship of the HeLa cell viability evaluated by MTT assay with various concentrations of PDMAEMA45-PMAEBA50-15 spheres, PDMAEMA45-PMAEBA50-20 nanorods, PDMAEMA45PMAEBA100-15 nanowires, and the PDMAEMA45-PMAEBA250-10 vesicles at pH = 7.4.

spheres, nanorods, nanowires, and vesicles without DOX up to the concentration of 1.2 mg/mL; thus, the four nano-objects composed of the diblock copolymers, PDMAEMA45-bPMAEBAn are of very low cytotoxicity. Figure 8 shows the cytotoxicities of free DOX and the four different DOX-loaded nano-objects. The free DOX displays a

Figure 8. Relationship of HeLa cell viability evaluated by MTT assay with various concentrations of the vesicles-DOX, nanorods-DOX, nanowires-DOX, and spheres-DOX at pH = 7.4.

dose-responsive curve different from those of the DOX-loaded nano-objects, especially the nanowires-DOX; for example, the DOX dose required for 50% cellular growth inhibition (IC50) evaluated by HeLa cells is 2.04 μg/mL for the free DOX, but for the nanowires-DOX, the IC50 is 58.05 μg/mL, and then the cell viability does not obviously decrease with increasing DOX. As we mentioned in the size measurements by TEM, the average length and the D of this nano-object are approximately 2000 and 20 nm, respectively, and their size in the solution should be bigger than that measured by TEM, so big size nanowires cannot be internalized by HeLa cells, which is consistent with the results reported.63 In testing cytotoxicities of the spheres-DOX, nanorods-DOX, and vesicles-DOX to the HeLa cells, we observed three different dose-responsive curves 18355


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Figure 9. Confocal laser scanning microscope images of the HeLa cells treated with free DOX (A), the spheres-DOX (B), the vesicles-DOX (C), the nanorods-DOX (D), and stained with DAPI and Lyso Tracker green DND (green) at 37 °C for 24 h for each panel. The images from left to right display DAPI (blue), DOX (red), Lyso Tracker green DND (green), and a merge of the three images. All the nano-object solutions contain the DOX of 0.1 μg/mL.

polymer of the four nano-objects at mild condition; their DLCs are 11.5% for the spheres-DOX, 13.9% for the nanorods-DOX, 16.2% for the vesicles-DOX, and 14.7% for the nanowiresDOX. The in vitro release tests of the DOX-loaded nanoobjects reveal that aromatic imine is stable under neutral conditions, which is expected for effectively avoiding premature drug release in blood circulation, and the release rate of DOX is faster in a solution at pH = 5.0 than that at pH = 6.0. The block copolymer nano-objects are of low cytotoxicity, and the DOXloaded nano-objects display their cytotoxicity in the following order: nanorods-DOX > vesicles-DOX > spheres-DOX. However, the endocytosis tests demonstrate that the internalization of nanorods-DOX is slower than that of the vesiclesDOX, but faster than that of the spheres-DOX, and the large size nanowires cannot be internalized by HeLa cells. Although all three nano-objects are mainly localized in the acidic organelles of the HeLa cells, the nanorods localized in the acidic organelles are relatively more than the vesicles-DOX, leading to faster intracellular release of DOX from the nanorods-DOX in comparison with the release from the

the triggered DOX release via the cleavage of imine linkage. Compared to the HeLa cells stained by Lyso Tracker green DND (green) in Figure 9B3−D3, the red fluorescence from DOX is mainly localized in the acidic organelles for all the three DOX-nano-objects, but, relatively, the nanorods-DOX localized in the acidic organelles are more than the vesicles-DOX as shown in Figure 9C2,4,D2,4. Thus, more DOX molecules release from the nanorods-DOX, leading to higher cytotoxicity of the nanorods-DOX in comparison with the vesicles-DOX.

4. CONCLUSION For understanding the influence of nano-objects’ morphologies on the control release of drug and better designing polymeric drug carriers, three canonical structures of spheres, nanorods, and vesicles with similar size and the large size nanowires have been successfully prepared by RAFTDP using PDMAEMA as macro-CTA, and for efficient conjugation of the drug onto the core polymer chains via acid-labile imine linkage, the functional monomer, MAEBA is used as a core monomer for the first time. The DOX is successfully conjugated onto the core 18356


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Figure 10. Cellular internalization analysis for three types of PDMEMA45-b-PMAEBAn assemblies and free DOX. (A) Overlay CLSM images of HeLa cells upon incubation with three types of distinct nanostructures (spheres, rods, and vesicles) and free DOX with varying time intervals. The nucleus were stained with DAPI (blue channel), and red channel fluorescence emission originated from DOX moieties. (B) Normalized fluorescence intensity (red channel) of HeLa cells was quantified from CLSM observations. All data are mean values (∼20 cells, three parallel experiments, P < 0.05). All nano-object solutions contain the DOX of 0.23 μg/mL. (6) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (7) Liu, S.; Maheshwari, R.; Kiick, K. L. Polymer-Based Therapeutics. Macromolecules 2009, 42, 3−13. (8) Phillips, M. A.; Gran, M. L.; Peppas, N. A. Targeted Nanodelivery of Drugs and Diagnostics. Nano Today 2010, 5, 143−159. (9) Kim, B. S.; Park, S. W.; Hammond, P. T. Hydrogen-Bonding Layer-by-Layer-Assembled Biodegradable Polymeric Micelles as Drug Delivery Vehicles from Surfaces. ACS Nano 2008, 2, 386−392. (10) Torchilin, V. P. Multifunctional Nanocarriers. Adv. Drug Delivery Rev. 2006, 58, 1532−1555. (11) Duan, X.; Xiao, J.; Yin, Q.; Zhang, Z.; Yu, H.; Mao, S.; Li, Y. Smart pH-Sensitive and Temporal-Controlled Polymeric Micelles for Effective Combination Therapy of Doxorubicin and Disulfiram. ACS Nano 2013, 7, 5858−5869. (12) Champion, J. A.; Katare, Y. K.; Mitragotri, S. Particle Shape: a New Design Parameter for Micro- and Nanoscale Drug Delivery Carriers. J. Controlled Release 2007, 121, 3−9. (13) Cho, E. J.; Holback, H.; Liu, K. C.; Abouelmagd, S. A.; Park, J.; Yeo, Y. Nanoparticle Characterization: State of the Art, Challenges, and Emerging Technologies. Mol. Pharmaceutics 2013, 10, 2093−2110. (14) Gratton, S. E. A.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.; DeSimone, J. M. The Effect of Particle Design on Cellular Internalization Pathways. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11613−11618. (15) Stolnik, S.; Illum, L.; Davis, S. S. Long Circulating Microparticulate Drug Carriers. Adv. Drug Delivery Rev. 2012, 64, 290−301. (16) Tang, L.; Gabrielson, N. P.; Uckun, F. M.; Fan, T. M.; Cheng, J. Size-Dependent Tumor Penetration and in vivo Efficacy of Monodisperse Drug-Silica Nanoconjugates. Mol. Pharmaceutics 2013, 10, 883−892. (17) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of Sub-100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat. Nanotechnol. 2011, 6, 815−823. (18) Liang, H. F.; Chen, C. T.; Chen, S. C.; Kulkarni, A. R.; Chiu, Y. L.; Chen, M. C.; Sung, H. W. Paclitaxel-Loaded Poly(gamma-Glutamic Acid)−Poly(lactide) Nanoparticles as a Targeted Drug Delivery System for the Treatment of Liver Cancer. Biomaterials 2006, 27, 2051−2059.

vesicles-DOX. Preparation of polymer prodrug nano-objects and morphology-tunable biological performance open a new insight for exploring new generation polymeric nano-objectsbased drug delivery systems with improved efficacy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04693. Characterizations of the polymers and the aggregates obtained (Figures S1−S8 and Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(C.-Y.H.) E-mail: [email protected]. *(C.-Y.P) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China under Contract Nos. 21074121, 21090354, and 21374107.

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REFERENCES

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Fabrication of Functional Nano-objects through RAFT Dispersion

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