Swollen Micelles for the Preparation of Gated, Squeezable, pH

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Swollen Micelles for the Preparation of Gated, Squeezable, pH-Responsive Drug Carriers Jian-Bo Qu, Robert Chapman, Fan Chen, Hongxu Lu, and Martina Heide Stenzel ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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ACS Applied Materials & Interfaces

Swollen Micelles for the Preparation of Gated, Squeezable, pH-Responsive Drug Carriers Jian-Bo Qu*,†,‡ Robert Chapman,‡ Fan Chen, ‡ Hongxu Lu,‡ Martina H. Stenzel*,‡ †

State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology,

China University of Petroleum (East China), Qingdao 266580, P.R. China ‡

Centre for Advanced Macromolecular Design (CAMD), School of Chemistry, University of New

South Wales, Sydney, NSW 2052, Australia *Corresponding author, Email: [email protected], [email protected]

ABSTRACT: Natural variations in pH levels of tissues in the body make it an attractive stimuli to trigger drug release from a delivery vehicle. A number of such carriers have been developed but achieving high drug loading combined with low leakage at physiological pH and tunable controlled release at the site of action is an ongoing challenge. Here we report a novel strategy for the synthesis of entirely hydrophilic stimuli responsive nanocarriers with high passive loading efficiency of doxorubicin (DOX), which show good stability at pH 7 and rapid tunable drug release at intracellular pH. The particles (Dh = 120-150 nm), are prepared by crosslinking the core of swollen

micelles

of

the

triblock

copolymer

poly[poly(ethylene

glycol)

methyl

ether

methacrylate-b-N,N’-dimethylamino methacrylate-b-tert-butyl methacrylate] (poly(PEGMEM A)-bPDMAEMA-b-PtBMA)). After subsequent deprotection of the tert-butyl groups a hydrophilic poly(methacrylic acid) (PMAA) core is revealed. Due to the negative charge in the acidic core the particles absorb 100% of the DOX from solution at pH 7 at up to 50% wt DOX / polymer, making them extremely simple to load. Unlike other systems, the DMAEMA ‘gating’ shell ensures low drug 1

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leakage at pH 7, while physical shrinkage of the MAA core allows rapid release below pH 6. The particles deliver DOX with high efficiency to human pancreatic cancer AsPC-1 cell lines, even lowering the IC50 of DOX. As the particles are stable as a dry powder and can be loaded with any mixture of positively charged drugs without complex synthetic or purification steps, we propose they will find use in a range of delivery applications.

KEYWORDS: swollen micelles, squeezing effect, gating, pH-responsive nanoparticles, doxorubicin

INTRODUCTION Over the past two decades a tremendous amount of effort has been directed towards the development of drug delivery systems, especially in cancer therapy.1–3 Such systems are able to prolong the circulation time and control the drug release rate at the target site, greatly reducing the amount of drug required and undesirable side effects on healthy tissue.4 A myriad of nanoparticles (NPs) have been developed for this purpose, such as micelles, liposomes, vesicles, dendrimers, polymer-drug conjugates and inorganic NPs.1,5,6 Stimuli-responsive nanocarriers which are able to release their cargo in response to changes in temperature, pH, magnetic field, the ionic strength of the solution, and or in the presence of enzymes, have shown great potential at improving the specificity of drug delivery.7–9 pH-responsive carriers have attracted particular interest since well-defined pH gradients exist in different organs, tissues and cellular compartments,10 such as in the gastrointestinal tract (pH 1-7.5),11 sites of inflammation and infection (pH 5.4-5.7),12 solid tumors

(pH 5.8-7.2),13–16

endosomes

and

lysosomes

(pH 4.5-5.5).15,17

2


Correspondingly,

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pH-responsive nanocarriers are usually designed to respond the pH gradients within the microenvironments of organs, tissues, and cell organelles.11 In order to achieve pH dependent drug release, the carrier must undergo a very sharp change in physical properties within a narrow pH window, and one or more of four release mechanisms are typically employed to achieve this. Either (i) the drug is covalently bound to the carrier via an acid-labile linkage (eg. hydrazine, ortho ester, imine, acetal) which is cleaved at lower pH values,6,18–21 or (ii) ionizable polymers are used to swell the outer shell or the core of the carrier,21–23 or (iii) ionizable polymers are used to destabilize the entire micelle / liposome,24–26 or (iv) the surface charge of the carrier switches under acidic conditions to affect the rate of cellular uptake.15,27–29 Regardless of the strategy, it is difficult to achieve both high loading of drug and low leakage at pH 7, while retaining a rapid controllable release at pH 5-6. Most systems are also synthetically complicated, requiring either complex chemistry to conjugate the drug, or lengthy purification steps after loading of the drug. The driving force for drug release is typically diffusion, which limits the rate of release according to Fick’s First Law. In this work we have designed a carrier that can be stored as a dry powder, loaded with very high quantities of drug without purification, is stable at physiological pH, and which releases the drug rapidly below pH 5.0 through a squeezing mechanism. There are a few examples of a pH driven ‘squeezing’ mechanism in the literature that inspire our system. Kim and coworkers first demonstrated this with C-25 gel. At pH 2.0 the gels were highly swollen, but at pH 7.4 the amine groups became deprotonated and shrunk dramatically, expelling the drug.30 In their work, the hydrogel was contained within a large porous rigid container and so was only suitable for controlled oral drug delivery. More recently, Bronich and coworkers used a similar design to prepare polymer 3



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micelles with cross-linked polyanion core for delivery doxorubicin (DOX).31 They used Ca2+ ions to drive the assembly a commercial poly(ethylene oxide)-b-(methacrylic acid) (PEO-b-MAA), and then used ethylene diamine to crosslink the PMAA core. At pH 7 the charged core enabled passive loading of as much as 40-100% (wt/wt) of DOX relative to the polymer. When the pH was reduced to 5.5, the core shrunk driving release of the drug, at a rate that could be tuned to some extent by the crosslinking density. However, because the polyanion core was swollen at pH7.4, these capsules were extremely leaky, and more than 60% of the loaded DOX was released within 48h, limiting the usefulness of this system. Herein we have used controlled polymerization to prepare hairy NPs with a cross-linked PMAA core and PEGMEMA outer shell, and have introduced a positively charged DMAEMA block between the two in order to improve stability at pH 7 through a gating mechanism. The synthesis follows

three

steps

(Scheme

1).

A

triblock

macroRAFT

agent

(poly(PEGMEMA)-b-PDMAEMA-b-PtBMA) was first prepared and assembled into micelles which were swollen with tBMA monomer and crosslinker (ethyleneglycol dimethacrylate, EGDMA). After further polymerization to chain extend the macroRAFT agent and crosslink the core tert-butyl group was removed to reveal a PMAA core. These particles represent the first drug nanocarriers that demonstrate both a squeezing and gating effect. DOX was chosen as a model anticancer drug owing to its effectiveness against a wide range of cancers, and was passively loaded into the nanoparticles via electrostatic interaction. The squeezing effect of the polyanion core and gating effect of the hairy shell was evaluated by the change in morphology, charge and DOX release rate from the NPs at pH’s from 5-7.4. Finally the in vitro cellular cytotoxicity studies were performed to evaluate the biological activity of DOX-loaded pH-responsive hairy NPs. 4


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Scheme 1. Preparation of hairy PMAA NPs with core-shell structure through swollen micelles via RAFT polymerization, and releasing mechanism of DOX from the NPs with squeezing and gating effects.

RESULTS AND DISCUSSION Synthesis of Triblock MacroRAFT Agent. Each block of the amphiphilic ABC triblock terpolymer was designed with a specific function in mind: The first block, poly(PEGMEMA), was chosen to increase the biocompatibility and stability of the NPs. The well-known ‘stealth’ effect of PEG is also known to improve circulation times in vivo.32 The second PDMAEMA block was 5



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chosen to provide a gating effect as the polymer is expected to form a dense layer in the deprotonated state and a swollen more layer when protonated under acidic conditions. The third PtBMA block, was chosen as the hydrophobic block to drive the self-assembly into micelles. This block was polymerized last block last, so as to allow simultaneous chain extension and crosslinking of the core of the micelles after swelling with tBMA and EGDMA. The labile protecting group of

tBMA allows hydrolysis of the core to PMAA after synthesis, which was expected to act as a sponge for the quick uptake of positive charged drug. In this study, three triblock macroRAFT agents with different PDMAEMA and PtBMA block lengths were prepared, as shown in Table 1. Block lengths were chosen so as maintain a constant hydrophilic-lipophilic balance (HLB) within the range of 12-18 necessary for stable micelles in water, at different sizes of PDMAEMA shell. The HLB was calculated from the ratio of the molecular weights of the hydrophilic segment (Mh) and the total polymer (M),33 and was measured to be 15.0 ± 0.3 for three polymers (see Table 1). All polymerizations were well controlled as evidenced by clean shifts in the GPC peaks within almost no tailing and low dispersities (Ð < 1.2, see Supporting Information, Figure S2). Preparation of Hairy PtBMA NPs Via RAFT Polymerization. Hairy PtBMA NPs were prepared by assembly of the triblock copolymer into micelles and subsequent chain extension in the swollen micelles with tBMA and the crosslinker EDGMA. For each polymer, swollen micelles with 10%, 20% and 30% (w/w) of crosslinker tBMA were prepared by ultrasonication method (Table 2). In order to keep the same density of hydrophilic brush on each particle, the amount of macroRAFT agent in each group was kept constant. A trace amount of sodium chloride was added to the water (0.01 wt%) to decrease the water solubility of the tBMA, and a small amount of hexadecane (2 wt%) was added to help stabilize the swollen micelles.34 From Table 2 we can find the number-average 6


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hydrodynamic diameter (dh) of all samples prior to the chain extension was 160-190 nm, and the droplet size distributions (Ð) were moderate. Colloidal stability of these swollen micelles was retained for at least 3 days. After assembly, the swollen micelles were degassed and polymerized, resulting in NPs with cross-linked PtBMA cores and poly(DMAEMA-b-PEGMEMA) shells (Scheme1), following the conditions shown in Table 2. The conversion of both monomer and cross-linker was 100% in all cases, as determined by 1H NMR (DMSO-d6) (see Supporting Information, Figure S3). Figures 1a-c show the number distribution as measured by dynamic light scattering (DLS) for the nine PtBMA NPs. The particle diameters were found to increase slightly after polymerization, possibly due to Ostwald ripening during the reaction, but the particle size distributions became narrower. Increased cross-linking density led to slightly larger particles, although this effect was very small.

Table 1. RAFT polymerization of triblock macroRAFT stabilizers.

CTA

Monomer

1 CPADB PEGMEMA

Mn,thb Mn,SECc

[M] / [CTA] / [AIBN]

Conv. (%)

DP

15:1:0.15

92

14

4400

22:1:0.1

74

16

40:1:0.1

78

a

Đc

HLB

5580

1.14

-

6950

7100

1.14

-

31

9300

8890

1.13

-

-1

(g mol )

2

1

3

1

4

1

80:1:0.1

66

53

12720

11100

1.11

-

5

2

20:1:0.1

80

16

9220

9560

1.13

15.1

6

3

30:1:0.1

79

24

12670

12050

1.09

14.7

7

4

40:1:0.1

75

30

17000

15540

1.13

15.0

DMAEMA

tBMA

Polymerizations were conducted at 65 ºC for 15-16 h. a Degree of polymerization determined by the 7



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conversion. b Calculated using the conversion obtained from 1H NMR. c Determined by DMAc GPC relative to PMMA standards without correction.

Table 2 Recipes of swollen micelles employed before polymerization Codea

a

CDb (%)

Oil phased

macroRAFTc (mg)

dh

PDI

tBMA(mg)

EGDMA(mg)

(nm)

207

23

158±3

0.185

184

46

178±8

0.132

D16-10%

10

D16-20%

20

D16-30%

30

161

69

170±5

0.140

D31-10%

10

207

23

163±5

0.157

D31-20%

20

184

46

170±3

0.173

D31-30%

30

161

69

181±6

0.175

D53-10%

10

207

23

161±3

0.210

D53-20%

20

184

46

167±6

0.169

D53-30%

30

161

69

162±2

0.203

18.2

25

33.5

all samples were named using DP of DMAEMA and the amount of added crosslinker (CD). b CD:

mass ratio of added crosslinker to the total amount of crosslinker and monomer. c 5 mL of water phase contained macroRAFT stabilizer and 0.01wt% NaCl. d The oil phase was comprised of tBMA, EGDMA, AIBN (0.5 times of [macroRAFT]), HD (2 wt%) and trioxane in toluene (12.5 mg).

In order to obtain pH-responsive NPs, the tert-butyl groups on the PtBMA NPs were removed by hydrolysis with trifluoroacetic acid. Figure 1d shows the FT-IR spectra of a representative set of PtBMA NPs before and after hydrolysis. In all samples a broad peak was observed at 3350 cm-1 8


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after hydrolysis, ascribed to the stretching vibration of the CO2H. In the spectra of 10% and 20% cross-linked particles, the characteristic peaks of the tert-butyl at 1392 cm-1 and 1367 cm-1 disappeared suggesting complete hydrolysis. In most highly cross-linked samples, a small peak at 1367 cm-1 was still observed even after 12 h of hydrolysis, suggesting significant but not complete removal of the tert-butyl groups in these cases. This is likely due to the complete conversion of crosslinker (Figure S3), which hinders the penetration of TFA into the core. These conditions resulted in no cleavage of the ester groups in the PEGMEA and DMAEMA blocks of an un-crosslinked triblock copolymer, as determined by NMR, suggesting no significant cleavage of these groups should be expected in the crosslinked particles either. After staining with uranyl acetate, the morphology of the hairy NPs was observed by transmission electron microscopy (TEM). Figure 2 shows the TEM images of the D31-20% PtBMA NPs before and after hydrolysis. The particles were close to spherical, roughly 60-100 nm in diameter, consistent with the DLS measurements. No apparent differences among the particles with different brush lengths and CDs were observed (Figure S4). Slight variations in color around the periphery of the particles is indicative of the different structure between the hydrophilic shell and cross-linked hydrophobic core (Figure 2 inset).

9



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Figure 1. Size distributions by number of PtBMA NPs with different crosslinking densities (CDs) (a-c), and FT-IR spectra of PtBMA NPs before and after hydrolysis (d).

Figure 2 TEM images of D31-20% hairy PtBMA NPs before (a) and after (b) hydrolysis. pH-Responsive Properties of Hairy PMAA NPs. The effect of pH on the morphology of the hairy PMAA NPs was initially investigated by measuring the zeta potentials and hydrodynamic diameter of the NPs in different 10 mM buffers. As shown in Figure 3a,b the zeta potential of the hairy

10


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pMAA NPs decreased with increasing pH, consistent with ionization of the carboxylic acid groups (from the PMMA core) and deprotonation of the tertiary amine groups (from the PDMAEMA shell). At low pH, where the core is expected to be protonated, the surface charge was found to be positive, but at pH’s greater than 6, all particles exhibited a negative surface charge due to collapse of the PDMAEMA shell, and the ionization of the dense PMAA core and α chain end acid groups on the shell. As the crosslinking density (CD) was increased in the PMAA NPs at any given DMAEMA content and pH, the zeta potential was also found to increase (Figure 3a and Figure S5). This is likely due to both a reduction in the absolute number of carboxylic acid groups in the core and the resistance to protonation of the acid groups (at the polymer α chain end and in the core) caused by the tighter packing of NPs that occurs at higher crosslinking density. The zeta potential also increased with increasing DMAEMA content at pH’s below its pKa of about 7.0 (Figure 3b) for any given CD.35 This trend is not present at higher pH’s, where the DMAEMA is largely deprotonated, and does not contribute to the overall zeta potential. The zeta potential was also determined in cell growth media. The tentative values for all samples were around -9 mV. However, the instrument reading was unreliable and prone to errors and the results are therefore not included. The increase in zeta potential may indicate the absorption of some proteins from the cell growth media. The particle swelling was also strongly affected by pH as shown in the hydrodynamic diameters reported in Figure 3c and 3d. At low pH, where ionization of carboxylic groups in the core is minimal, small particles were observed. Increasing the pH lead to increased electrostatic repulsion in the network and swelling of the particle. As expected particles with lower CD presented the greatest swelling as these networks are less tightly bound and more readily deprotonated (Figure 3c). Similar trends were observed for the two other groups of PMAA NPs (D16 and D53, Figure S6). The 11



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diameter of NPs with the same CD’s increased with increasing DMAEMA content at any given pH, but showed similar overall amounts of swelling (Figure 3d). It should be noted that the swelling of the PMAA core with increasing pH will actually be larger than the observed size change for the entire NP, as it will be somewhat offset by the shrinking DMAEMA shell.

Figure 3 Zeta potentials (a, b) and number-average diameters (c, d) of hairy PMAA NPs as a function of pHs. The concentration of pH buffer is 10 mM.

DOX Loading and Release. The size distribution of the NPs was found to be unaffected by freeze-drying subsequent rehydration of the dried powder (Figure S7). As a result this system can be stored for long periods of time and easily loaded with any positive drug (or mixture of drugs) by rehydration in the desired solution at the clinic. In order to demonstrate the potential for this system 12


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we incorporated DOX into the PMAA NPs by passive loading and studied the loading efficiencies, stability at physiological pH and release at reduced pH. By using DOX we were able to compare our system to those already reported in the literature, and for this reason we also performed the loading at pH 7.0.31 As shown in figure 4a the drug loading capacity of the D53-10% PMAA NPs was found to increase with the concentration of DOX to a maximal DOX loading content of around 745 µg/mg NPs (74.5% w/w). Remarkably, the percentage of drug absorbed by the particles from solution (which we defined as the drug loading efficiency) was approximately 100% at loadings as high as 584 µg/mg (58.4 % w/w). Thus, we find that not only do these particles exhibit very high loading capacity, but they also show almost complete uptake of the drug eliminating the need to even purify the particles. As shown in Figure 4b, the DOX loading content of particles decreased with increasing CD, which we attributed to the reduced number of deprotonated carboxylic acid units. The length of the PDMAEMA-b-poly(PEGMEMA) brushes had no significant effect on the DOX loading content. While other systems have achieved loading efficiencies in the range we report, they tend to be highly leaky (and therefore toxic) even at neutral pH. For this reason we compared the release profiles of DOX-loaded hairy PMAA NPs at the three different CDs at both pH 7.4 and 5.0, as shown in Figure 4c. It is evident that DOX release from the PMAA NPs was a pH-dependent process (Scheme 1), and the release rate from all samples was much faster at pH5.0 than at pH7.4 due to the disappearance of electrostatic interaction between NPs and DOX at this pH. However, the variation of DOX release rate at pH 5.0 and pH 7.4 was different for PMAA NPs with different CDs, which we ascribe to different squeezing effects of the PMAA cores. The NPs with medium CD (D31-20%) were found to exhibit the best squeezing effect. Particles with a lower CD (D31-10%) 13



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were not able to shrink sufficiently to push the drug out, while particles with higher CD (D31-30%) did not swell enough in first place in order to display an efficient squeezing effect. The swelling is governed by repulsion of the negative charges, which leads to the elastic stretching of the polymer, a process that is controlled by the crosslinking density. The entropic elasticity functions like a spring that releases its pressure rapidly when the polymer becomes protonated, snapping the chains back to an entropically favorable conformation. The release profiles in figure 4c demonstrate a rapid release over the first 6 h followed by a slower release stage up to 36 h and, in the case of the D31-20% PMAA NPs, were around three times faster at pH 5.0 than at pH 7.4. Only 25% of the encapsulated DOX was released from the particles at pH 7.4 in the first 6 h, compared to nearly 75% at pH 5.0. Similar cross-linked PEO-b-PMAA micelles have been reported to release over 60% of their loaded DOX within 48h at pH 7.4,31 which is highly problematic for any triggered drug release application. In this study the release of DOX from hairy PMAA NPs was less than 36% within 36 h. The slower rate of DOX release at pH 7.4 is driven by the gating effect of poly(PEGMEMA)-b-PDMAEMA brushes on the particle shell. At pH 7.4 the positively charged PDMAEMA chains will strongly bind to the negative charged PMAA core through electrostatic interaction, the release of DOX from the particles will be inhibited (Scheme 1). In order to examine the effect of brush lengths on the gating effect, and therefore the effect of the PDMAEMA length, we investigated the DOX release from PMAA NPs with same CD and different PDMAEMA lengths. Interestingly particles with medium DMAEMA chain lengths showed the greatest difference in release rate between pH7.4 and pH5.0. (Figure 4d). NPs with short PDMAEMA chains were unable to shield the surface of particles sufficiently at pH7.4, leading to an undesirable fast release of DOX. NPs with longer PDMAEMA chains were able effectively inhibit the release of DOX at pH7.4 but 14


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also inhibited the release of DOX at pH 5.0, presumably because the longer brush length increases the diffusive resistance of DOX from the particles. PMAA NPs with the intermediate brush length and CD were able to inhibit release at pH 7.4, while maintaining rapid release at pH 5.0, and these particles were taken forward to in vitro testing.

Figure 4 (a) DOX loading content and efficiency of D53-10% PMAA NPs at different concentrations of DOX, (b) DOX loading content of PMAA NPs with different CDs and brush lengths (initial DOX concentration, 600µg/mL), (c) release profiles of DOX from PMAA NPs with different CDs at 37 ºC. D31-10% (■) pH5.0, (□) pH7.4; D31-20% (●) pH5.0, (○) pH7.4; D31-30% (▲) pH5.0, (△) pH7.4, and (d) release profiles of DOX from PMAA NPs with different brush lengths at 37 ºC. D31-20% CD (●) pH5.0, (○) pH7.4; D16-20% (■) pH5.0, (□) pH7.4; D53-20% (▲) pH5.0, (△) pH7.4. Here pH5.0 is acetate buffer (0.14M NaCl) and pH7.4 is PBS buffer. Cellular Uptake and Cytotoxicity Against AsPC-1 Cells. Human pancreatic carcinoma (AsPC-1) cell lines were chosen as a modal system to investigate the ability of our NPs to transport drug into the cytoplasm. While DOX is easily uptaken by these cells without a carrier this system allows easy 15



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comparison of our NP carriers to the existing literature. A confocal (CLSM) study was first carried out to observe the uptake of DOX-loaded hairy PMAA NPs with different CDs and brush lengths by AsPC-1 cells (Figure 5). An intense green fluorescence signal was observed in all cells and the overlap of this signal with the dye labeled lysosomes indicates the mechanism of uptake was clathrin-mediated endocytosis.36 In addition, a higher magnification imaging under confocal microscopy revealed that the green DOX was partially overlapped with the blue cell nucleus which indicates that the DOX had been released into the cell nucleus (Figure S10). The fluorescence intensity of cells after incubation with D31-30% PMAA NPs is the strongest among the five samples we studied, despite these particles having the least amount of DOX (Figure 4b), indicating these NPs were most readily internalized by AsPC-1 cells. While many variables such as size, shape, and surface charge, are known to impact nanoparticle uptake into cells,36 in this study there was no difference in the morphology of all NPs. The more positive zeta potential of the D31-30% PMAA NPs at pH 7 (Table S1) could be the source of the increased uptake rate in this case. However, the possibility of the formation of a protein corona needs to be considered and there is a change that these nanoparticles attract different proteins that will influence the uptake. Another probable reason is that the cores of D31-30% PMAA NPs with the highest crosslinking degree have the highest mechanical strength among the five samples. We further studied the relative rate of NP internationalization for particles with varying DMAEMA brush lengths at 20% CD by flow cytometry (Figure S8, Table S2, S3). After 3 h of incubation, no significant difference in the cellular internalization of all NPs was observed, confirming the efficiency of uptake regardless of PDMAEMA length. The reason for this probably lies in their similar particle sizes and surface charges (Table S1). 16


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Figure 5. CLSM images of AsPC-1 Cells after incubation with DOX-loaded hairy PMAA NPs for 2 h at 37 ºC. Green fluorescence represents DOX; red fluorescence represents lysosomes stained with Lyso Tracker Red DND-99.

We then proceeded to study the cytotoxicity of loaded, and non-loaded NPs against the AsPC-1 cell line. Empty NPs did not show any toxicity towards the cells (Figure 6a), but actually increased cell viability at higher NP concentrations, indicating the blank particles were able to traffic nutrients into the cells and stimulate their growth. Because free DOX is able to penetrate the cell membrane so efficiently, many studies find that loading the drug in a nanocarrier will actually reduce the toxicity of the drug.31,37–40 By contrast in our case, the IC50 of the DOX-loaded NPs was lower than 17



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Page 18 of 31

free DOX (Table 3 and Figure S9), indicating the power of this combined gating and squeezing approach not only to enable high loading, and low leakage, but also high toxicity of the drug at the site of action. At a DOX concentration of 2.5 µM (close to IC50 of free DOX), the cytoxicity of loaded D31-20% was slightly higher than the free DOX and the loaded D16-20% and D53-20% particles (Figure 6b). This further confirms the gating and squeezing advantages of the intermediate DMAEMA brush length and intermediate crosslinking density.

Figure 6. Cytotoxicity of free hairy PMAA NPs (a), DOX-loaded hairy PMAA NPs and free DOX (b) against AsPC-1 cells after 72 h.

Table 3 IC50 of DOX-loaded hairy PMAA NPs to As-PC 1 cells and the corresponding polymer concentration Code

DOX loading content (µg/mg)

IC50 (µM)

Polymer con. at the IC50 (µg/mL)

D16-20%

410.6

1.63±0.12

2.16

D31-20%

424.7

1.68±0.44

2.15

D53-20%

434.4

2.05±0.71

2.57

Free DOX

/

2.23±0.37

18


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CONCLUSIONS In this study we have demonstrated the design of a novel pH-responsive drug delivery system with a core-shell structure. The particles have a high capacity for passive loading of positively charged drugs such as DOX at pH 7.0 (up to 74.5% w/w DOX / polymer), allowing them to be loaded from dry powder without purification. The cross-linked anionic cores result in a squeezing effect at pH 5.0 resulting in quick release of the drug, and the positive charged DMAEMA brushes gate the core at pH 7.4 resulting in low leakage during circulation. The CDs and brushes length were optimized to give the best release profiles of DOX. High cellular uptake of the carriers, and rapid release of the drug resulted in enhanced cytotoxicity of DOX (IC50) towards AsPC-1 cells, in contrast to many of the delivery systems reported to date. This platform is therefore not only simple to load with a range of drugs and highly stable at physiological pH, but also highly toxic once endocytosed thanks to this novel combination of squeezing and gating effects.

EXPERIMENTAL Materials. All chemicals were reagent grade and used as received, unless otherwise stated. Trifluoroacetic acid (TFA, 99%, Aldrich), 1,3,5-trioxane (99%, Aldrich), hexadecane (HD, 99%, Aldrich), toluene (99%, Ajax), n-hexane (>95%, Ajax), dimethylacetamide (DMAc, 99.9%, Aldrich), dimethyl sulfoxide (DMSO, 99%, Ajax), doxorubicin hydrochloride (DOX, ≥98%, Cayman

Chemical

Company),

Cyanine5

amine

(Cy5,

Lumiprobe,

USA),

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC.HCl, 98%, Fluka) and 4-(dimethylamino)pyridine (DMAP, 99%, Aldrich) were used as received. Deuterated NMR solvents

(CDCl3,

DMSO-d6)

were

purchased

from

Cambridge

19


Isotope

Laboratories.


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2,2’-Azobis(isobutyronitrile) (AIBN, 98%, Fluka) was recrystallized twice from methanol before use. N,N’-Dimethylamino ethyl methacrylate (DMAEMA, 98%, Aldrich), tert-butyl methacrylate (tBMA, 98%, Aldrich ), ethylene glycol dimethacrylate (EGDMA, 98%, Aldrich) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Mn=300 g.mol-1, 98%, Aldrich,) were purified by passing through a column packed with activated basic alumina (50-200 µm, Scharlau) to remove the inhibitor. 4-Cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to a literature.41 Milli-Q water (>18.6 MΩ.cm) was produced by a Milli-Q osmosis system. Analyses. NMR spectroscopy was performed using a Bruker Avance 400 (400.13 MHz, 1H; 75.5 MHz, 13C), with a BBFO probe, and spectra were processed using the Bruker TOPSPIN 3.0 software. Gel permeation chromatography (GPC) was performed using dimethylacetamide (DMAc) + 0.01 % (w/v) LiBr as the eluent on a Shimadzu modular system comprising an auto injector, a Phenomenex 5.0 µm bead size guard column (50 × 7.5 mm) followed by three Phenomenex 5.0 µm bead-size columns (105, 104 and 103 Å), and a differential refractive index detector and a UV detector. Molecular weights were estimated relative to narrow molecular weight distribution poly(methyl methacrylate) (200 to 1x106 g.mol-1) calibration standards without correction. Dynamic light scattering (DLS) measurements were run on a Malvern Zetasizer Nano ZS (laser, λ=632 nm, angle=173°) to determine the sizes and zeta potentials of particles. Disposable cuvettes were used for the measurement of hydrodynamic diameter (Dh) and folded capillary cells for zeta potential measurements. Transmission electron microscopy (TEM) images were obtained using a JEOL 1400 TEM at an accelerating voltage of 100 kV. TEM samples were prepared by drop casting the emulsion on a carbon coated formvar copper grid. The grids were air dried and negatively stained with uranyl acetate (1% w/v, 2 min). Fourier transform infrared (FT-IR) spectra were recorded using 20


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an ALPHA spectrometer equipped with a versatile high throughput platinum ATR (Bruker). Varian Cary 300 UV-Visible Spectrophotometer (UV-vis) was used to determine the concentration of DOX at the absorbance of 485 nm. Three calibration curves of DOX were made at pH7.0 phosphate buffer (10mM), pH7.4 PBS (0.14M NaCl) and pH 5.0 acetate buffer (0.14M NaCl), respectively. A confocal laser scanning microscopy (CLSM) (LSM 780, Zeiss) was used to observe the internalization of pH-responsive nanoparticles in AsPC-1 cells. The Argon and DPSS 561-10 laser sources were used to excite the fluorescence of DOX and LysoTracker Red DND-99, respectively. The corresponding fluorescent images at 535–590 nm and 590-630 nm were taken by CLSM. The Zen2012 imaging software (Zeiss) was used for imaging acquisition and processing. Flow cytometer (FACSCanto II, BD Biosciences) was used to study the relative particles number internalized by AsPC-1 cells. All the experiments were performed at least in triplicate except TEM and CLSM observations. Synthesis of Triblock MacroRAFT Agent. poly(PEGMEMA)-b-PDMAEMA-b-PtBMA was synthesized in three steps via RAFT polymerization (Figure S1). RAFT polymerization of PEGMEMA. PEGMEMA (9 g, 0.03 mol), RAFT agent CPADB (0.559 g, 2×10-3 mol) and initiator AIBN (0.0493 g, 3×10-4 mol) were dissolved in toluene (20 ml) with 1,3,5-trioxane

(0.05

g)

as

internal

standard

to

give

a

ratio

of

[monomer]:[RAFT]:[initiator]=15:1:0.15. The mixture was thoroughly purged with high purity nitrogen for 30 min at 0 ºC. The polymerization was then carried out in an oil bath at 65 ºC for 16 h. After polymerization, the polymer was precipitated in hexane and dried under vacuum to yield poly(PEGMEMA) as a viscous red liquid. Chain extension of poly(PEGMEMA) with DMAEMA. MacroRAFT agent poly(PEGMEMA) (2.2 21



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Page 22 of 31

g, 5×10-4 mol), DMAEMA (1.729 g, 1.1×10-3 mol), AIBN (0.00821 g, 5×10-5 mol ) were mixed together in toluene (7.33 ml) to give a ratio of [monomer]:[RAFT]:[initiator]=22:1:0.1. The mixture was purged with high purity nitrogen in an ice bath for 30 min and then polymerized at 65 ºC for 15 h. The reaction was stopped by quenching with air. The solution was precipitated in hexane to yield a viscous red liquid. The two other polymers were prepared by the same procedure using [monomer]:[RAFT]:[initiator] ratios of 40:1:0.1 and 80:1:0.1. Chain

extension

of

poly(PEGMEMA)-b-PDMAEMA

with

tBMA.

The

poly(PEGMEMA)-b-PDMAEMA were further chain extended with tert-butyl methacrylate (tBMA). In a typical reaction, poly(PEGMEMA)-b-PDMAEMA (Mn,th=6953 g mol-1, 3.46 g, 4.98×10-4 mol) was mixed with tBMA (1.416 g, 9.96×10-3 mol) and AIBN (0.00818 g, 4.98×10-5 mol ) in toluene (6.64 ml) ([monomer]:[RAFT]:[initiator]=20:1:0.1). The solution was degassed with high purity nitrogen for 30 min at 0 ºC. After polymerization at 65 ºC for 16 h, the final product was obtained by precipitation in hexane to yield the product as a brittle pink solid. The two other triblock macroRAFT

stabilizers

were

synthesized

by

the

same

procedure

using

[monomer]:[RAFT]:[initiator] ratios of 30:1:0.1 and 40:1:0.1. Preparation of the pH-Responsive Hairy NPs. Three groups of hairy NPs were prepared from the poly(PEGMEMA)-b-PDMAEMA-b-PtBMA micelles solution. In a typical procedure poly(PEGMEMA)-b-PDMAEMA-b-PtBMA (Mn,th=9220 g.mol-1, 18.2 mg) and sodium chloride (NaCl, 0.5 mg) were dissolved in Milli-Q water (5 mL) in a 25 mL glass vial to prepare micelles firstly. In an eppendorf tube, tBMA (207 mg), EGDMA (23 mg), HD (7.5 mg), AIBN (0.162 mg, 0.5 equiv relative to polymer) and 1,3,5-trioxane (300mg/mL in toluene, 12.5 mg) were mixed and subsequently added to the micelles solution. The mixture was ultrasonicated using a digital sonifier 22


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(Branson 450, 70% amplitude, 5 mm tip diameter) for 10 min to facilitate the swollen process of micelles in an ice bath. The resulting swollen micelles was then transferred into a 10 mL glass vial with a stir bar, degassed with high purity nitrogen for 30 min in an ice bath and polymerized at 75 ºC for 18 h. DLS and TEM analyses were carried out on the diluted emulsion solution (0.03 mL of emulsion solution in 1 mL of Milli-Q water). The emulsion was purified by dialysis against 70% ethanol solution and then against Milli-Q water, and then lyophilized (Labconco FreeZone 6 plus, USA) to yield the PtBMA NPs as a white powder. In order to obtain pH-responsive hairy PMAA NPs, the tert-butyl protecting groups were removed by dissolving dry PtBMA NPs (90 mg) in 5 mL of TFA/H2O (9:1, v/v) in a 20 mL glass vial. After stirring at 25 ºC for 7 h, the mixture was dialyzed against Milli-Q water for 2 days (MWCO 3500), and lyophilized to obtain hairy PMAA NPs. DOX Loading and Release. DOX was loaded into hairy PMAA NPs via electrostatic interaction. Typically, PMAA NPs (5 mg) were mixed with 5 mL of DOX solution (200 µg/ml) in pH7.0 phosphate buffer (10mM) at 25 ºC and incubated for 21h. The residual DOX was removed by ultrafiltration using Amicon Ultra-15 centrifugal filter devices pretreated with DOX (MWCO 10,000 Da, Millipore). The concentration of DOX in the filtrate was determined by absorbance at 485 nm, and the drug loading content (DLC) was calculated from the mass balance of DOX. The drug loading efficiency (DLE) was calculated according to the following equation: DLE = (DOX added initially − DOX in filtrate after ultrafiltration)/(DOX added initially) × 100%. The release of DOX from the hairy PMAA NPs was carried out in PBS buffer (20 mL, pH7.4, 0.14M NaCl) and acetate buffer (20 mL, pH5.0, 0.14M NaCl) at 37 ºC by dialysis method using a membrane tubing (MWCO 3500 Da). 1.6 mL of DOX-loaded NPs was placed into membrane tubing and 0.6 mL of release medium was taken at selected time intervals. Each time 0.6 mL of 23



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fresh release medium was added to replenish the release system to maintain a constant volume. The concentration of DOX in the dialysate was measured by absorbance at 485 nm relative to a calibration curve in the same buffer. Cellular Uptake and Cytotoxicity Studies. Cellular uptake study. Human pancreatic carcinoma AsPC-1 cells were cultured in T-25 tissue culture flasks with RPMI1640 medium supplemented with 10% fetal bovine serum and antibiotics at 37 ºC and 5% CO2. Once the cells reach confluence, the cells were harvested by trypsin/EDTA treatment and resuspended in the medium for further evaluation. For the observation of cellular uptake of NPs with CLSM, AsPC-1 cells were seeded in 35 mm Fluorodishes at a density of 2 × 105 cell per dish and incubated for 2 days before the NPs were loaded into the dishes. The working concentration of NPs was 100 µg/mL and the incubation time was 3 h. The cells were then washed three times with Hank's Balanced Salt Solution (HBSS) and stained with LysoTracker Red DND-99 (100 nM in HBSS) for 1 min. After rinse with HBSS, the cells were mounted in HBSS and observed by CLSM. The Argon and DPSS 561-10 laser sources were used to excite the fluorescence of DOX and LysoTracker Red DND-99, respectively. The images were captured and processed with Zen 2012 software. The relative cellular uptake of nanoparticles was also evaluated with flow cytometry. 2 × 105 AsPC-1 cells were first seeded into each well of 6-well culture plates and cultured for 48 h. The cell culture medium was then replaced with new medium containing 100 µg/ml of Cy5-labeled PMAA NPs (see detail in Supporting Information) and incubated at 37 ºC. After incubation for 3 h, the cells were extensively washed with PBS (pH 7.4) to remove the residual adhering NPs, resuspended, and analyzed on a BD FACSCanto II flow cytometer. A total of 15,000 cells were analyzed in each group using the fluorescent signal on channel of FL1-H. 24


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Cellular cytotoxicity study. AsPC-1 cells were seeded in 96-well plates at a density of 4,000 cells per well with 100 µL of culture medium for 24 h. The sample solution to be tested was sterilized via UV irradiation (15 min) before the solution was serially halved via dilution in sterile Milli-Q water. The DOX-loaded NPs solutions were then loaded into the plate at 100 µL per well. After incubation for 72 h, supernatant was discarded and the plates were washed once with PBS. Then 100 µL of fresh warm medium with 5 µL WST-1 were added to each well and incubated for 2 h in the incubator. The absorbance of each well was read on a Bio-Rad BenchMark microplate reader at 440 nm with a reference wavelength of 650 nm. The cells treated with normal medium were used as controls. The date was analyzed and plotted with Graphpad Prism 6.0. ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publications website. DLS measurements; NMR spectra; additional TEM images; Zeta potentials; flow cytometric analysis; cell viability profiles; CLSM images; and synthesis scheme of triblock macroRAFT stabilizer. ACKNOWLEDGEMENTS We would like to thank the financial support of Australian Research Council (ARC), China Scholarship Council (No. 201506455008), and National Natural Science Foundation of China (No. 21176257). REFERENCES (1)

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TOC Graphic

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Swollen Micelles for the Preparation of Gated, Squeezable, pH

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