Nanoprecipitation of PHPMA (Co)Polymers into Nanocapsules

Letter pubs.acs.org/macroletters

Nanoprecipitation of PHPMA (Co)Polymers into Nanocapsules Displaying Tunable Compositions, Dimensions, and Surface Properties Xibo Yan,*,†,‡,§ Ricardo Ramos,†,‡,§ Elsa Hoibian,∥ Christophe Soulage,∥ Pierre Alcouffe,†,‡,§ François Ganachaud,†,‡,§ and Julien Bernard*,†,‡,§ †

Université de Lyon, Lyon, F-69003, France INSA-Lyon, IMP, Villeurbanne, F-69621, France § CNRS, UMR 5223, Ingénierie des Matériaux Polymères, Villeurbanne, F-69621, France ∥ Univ-Lyon, CarMeN laboratory, INSERM U1060, INSA Lyon, INRA U1397, Université Claude Bernard Lyon 1, F-69621 Villeurbanne, France ‡

S Supporting Information *

ABSTRACT: A series of PHPMA homopolymers and of mannose- and dimethylamino-functionalized copolymers, were prepared by RAFT polymerization and engaged in the preparation of oil-loaded nanocapsules using the “Shift’N’Go” process. Playing with the phase diagrams of both oil and homo- or copolymers afforded the preparation of functional camptothecin-loaded nanocapsules displaying tunable dimensions (90−350 nm), compositions and surface properties.

O

In this context, the one-step construction of precisely defined NCs from PHPMA homopolymers and its derivatives would be a significant progress in the field. Among different techniques, nanoprecipitation (i.e., supersaturation of a solute by solvent shifting) is a powerful method to produce colloids.9 The simplicity of the technique and the diversity of the resulting materials give impetus to design powerful nanocarriers for drug delivery systems.10 Recently, we derived the conventional nanoprecipitation process (of one solute) to generate functional NCs (made of two solutes) in a straightforward manner. What we called the “Shift’N’Go process” entails the simultaneous nanoprecipitation of an oil and a polymer, the latter which is simultaneously cross-linked to generate a durable shell. A careful establishment (and understanding) of both oil’s and polymer’s phase diagrams is the only requirement to generate precisely defined NCs.11 Previously limited to the Ouzo region of oil, we have demonstrated lately that the Shift’N’Go process also produces capsules in the “monophasic” domain, called Surfactant Free MicroEmulsion (SFME), in the upper part of the phase diagram.12 Monodisperse, thermodynamically stable sub-100

wing to their applications in the controlled delivery of pharmaceuticals, polymer-based nanocapsules (NCs), in which a cargo is confined in a cavity core protected by a polymeric shell, have attracted considerable attention lately.1 Such nanocontainers directly benefit from the properties of polymers for increasing drug efficacy, reducing toxicity and controlling biodistribution.2 However, their translation to in vivo administration is often hampered by the use of synthetic immunogenic polymers for shell construction. Poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) is a water-soluble, synthetic, vinyl-based polymer with singular nonimmunogenic and nontoxic characters. It has been broadly exploited for biomedical applications, to the extent that several PHPMA-based systems entered Phase I or II clinical trials for cancer chemotherapy applications.3 The presence of side-chain hydroxyl functions also makes PHPMA a perfect platform for incorporating diverse functional groups promoting biorecognition, cell-uptake, biodegradability, or bioimaging (e.g., carbohydrates, proteins, antibodies, fluorescent tags).4 Next to that, significant attention has been paid to the construction of PHPMA-based self-assemblies for biologically relevant purposes.5 In addition to micelles or polyplexes,6 few examples of polymersomes or hollow particles have been reported, relying either on the multistep synthesis of amphiphilic PHPMA-based macromolecules7 or the use of a sacrificial template approach.8 © XXXX American Chemical Society

Received: February 9, 2017 Accepted: March 27, 2017

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DOI: 10.1021/acsmacrolett.7b00094 ACS Macro Lett. 2017, 6, 447−451

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ACS Macro Letters nm nanodroplets can be selectively built and used as templates for the preparation of polymeric capsules (Scheme S1). In this communication, we report on the one-pot construction of precisely defined, oil-filled, PHPMA polymerbased NCs. We show that such NCs can be efficiently loaded with a hydrophobic drug in the course of the nanoprecipitation process, and the polymer shell designed to undergo degradation in reductive environments. To broaden the function of PHMPA-based capsules, we further generate NCs from a series of PHPMA copolymers functionalized with hydrophilic moieties (e.g., from a carbohydrate and/or a tertiary aminefunctionalized monomer(s), see Scheme 1). We show that the composition, dimension, and surface properties of the resulting NCs can be tuned in an extremely simple manner.

Figure 1. TEM images of (A) PHPMA259, (B) PHPMA322, and (C) PHPMA409 based NCs; (D) Fluorescence spectra of free and loaded camptothecin in water; (E, F) Degradation of cystamine cocrosslinked capsules by DTT.

Scheme 1. PHPMA Homo/Copolymers Prepared in This Study

HD concentration) and increase with acetone mass fraction. As nanoprecipitations are performed in similar composition ranges, the resulting NCs all show sizes of around 200 nm, according to TEM. DLS measurements confirmed the size of capsules in the acetone/water mixtures, with diameters of ∼200 nm and dv/dn below 1.20. After evaporation of acetone, diameters slightly increase (∼250 nm) owing to the swelling of polymer shell in water. To highlight the potential of PHPMA-based NCs, campthothecin, a hydrophobic anticancer drug, was preincorporated in acetone before solvent shifting. Fluorescence titration showed that the drug is successfully loaded within the liquid core of the NCs with an encapsulation efficiency of about 57% (Figure 1D and Supporting Information). Degradable NCs were also easily built using cystamine, predissolved in water, as a co-crosslinker together with IPDI. Owing to the presence of disulfide links within the cross-linking nodes, NCs vanished upon addition of dithiothreitol (DTT) as a reducing agent (see Figures 1E,F and S8). To modulate the surface properties of PHPMA-based capsules, we subsequently intended to introduce new functionalities into the PHPMA polymer chains prior to capsule fabrication. Owing to their significant role in cellular communication, biological recognition, and signal transduction,14,15 the incorporation of carbohydrate species into PHPMA backbones was investigated.15 A mannoside-based monomer, N-[2-(α-D-mannopyranosyl-oxy)ethyl] methacrylamide) (EMM) was selected, since its corresponding homopolymer exhibits no cytotoxicity (at 1 μM with intestinal epithelial T84 cells).16 EMM was randomly RAFT copolymerized with HPMA at different molar compositions ([HPMA]0/[EMM]0 = 9/1, 8/2, 7/3) targeting a final degree of polymerization ∼300. The three resulting HPMA copolymers, P(HPMA302-coEMM 35 ), P(HPMA 258 -co-EMM 80 ) and P(HPMA 228 -coEMM109) were characterized thoroughly (Table S1). The phase diagrams of the copolymers in water/acetone mixtures (Figure 2) highlight that the incorporation of mannose units significantly increases the “acetonophobic” character of PHPMA-based copolymers, as less acetone is required to precipitate the polymers (Wacetone from 0.75 to 0.66). Incorporation of 10 mol % of EMM exclusively enables the preparation of NCs in the SFME region (WHD = 0.05 wt %, WPHPMA = 0.05 wt %, and Wacetone = 0.75, diameter ∼208 nm,

For the core of the capsules, we used hexadecane (HD) as the oil, whose ternary phase diagram with acetone and water has been previously established (see Scheme S1).11a For the polymer shell, a series of PHPMA homopolymers were first synthesized by RAFT polymerization mediated by 4-cyano-4(phenyl-carbonothioylthio) pentanoic acid in DMSO/H2O mixture ([M]0/[CTA]0 = 400/1, 500/1, 800/1).13 In brief, HPMA was polymerized at 70 °C, using three different [HPMA]0/[CPADB]0 ratios, to yield PHPMAs exhibiting molar masses of 37.4, 46.3, and 58.8 kg mol−1 and Đ values below 1.3 (PHPMA259, PHPMA322, and PHPMA409, see Table S1). To overcome toxicity issues, the dithiobenzoate chain end was systematically removed through aminolysis. The phase diagrams of polymers in water/acetone mixtures were first carefully established following the procedure described in our previous work (see Supporting Information and refs 11 and 12). From these phase diagrams (Scheme S1), it appeared that the water-soluble PHPMA homopolymers actually require large amounts of acetone to precipitate; the cloud-point boundary curves were found at 85, 82, and 79 wt % for PHPMA259, PHPMA322, and PHPMA409, respectively. As these limits are well above the Ouzo domain identified for the HD/water/ acetone system (see Scheme S1), the preparation of HD-loaded PHPMA-based NCs can exclusively be envisioned in the SFME domain.12 To prepare the capsules, HD (0.05 wt %) was dissolved in acetone with the water insensitive cross-linker isophorone diisocyanate (IPDI, 0.005 wt %), whereas the PHPMA (0.05 wt %) was solubilized in water. The addition of the aqueous solution into the organic phase was then performed in a oneshot process with gentle mixing, right at the edge of the boundary curves (vide supra and Scheme S1). The IPDI rapidly reacts with hydroxyl moieties of PHPMA chains to gradually cross-link the polymeric shell. Core−shell structure of HD droplets surrounded by a polymer corona is given in Figure 1. As previously reported,12 in the SFME domain, the dimensions of HD droplets rely on the acetone/water ratio (rather than on 448

DOI: 10.1021/acsmacrolett.7b00094 ACS Macro Lett. 2017, 6, 447−451

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ACS Macro Letters

Figure 2. (Center) Overlapped phase diagrams of P(HPMA-co-EMM) copolymers and HD. TEM images of P(HPMA302-co-EMM35)-based NCs built in the SFME domain (A), of P(HPMA258-co-EMM80)-based NCs built in the SFME (B) and Ouzo (C) domains and of P(HMPA228-coEMM109)-based NCs built in the SFME (D) and Ouzo (E) domains. Horizontal lines in the phase diagrams correspond to the cloud-point boundaries of P(HPMA302-co-EMM35) (light blue line), P(HPMA258-co-EMM80) (dark blue line) and P(HPMA228-co-EMM109) (dark green line).

dv/dn = 1.20, Figure 2A) whereas further enrichment in mannose content results in enlargement of the manipulation region, with possibilities to generate NCs in both HD’s Ouzo or SFME domains. When processed in the SFME region (WHD = 0.05 wt % and WPHPMA = 0.05 wt %), mannose-functionalized NCs with diameter of 155 nm (Wacetone = 0.69) and 91 nm (Wacetone = 0.66), as given by DLS, were built with P(HPMA258co-EMM80) and P(HPMA228-co-EMM109), respectively. These sizes are in consistency with TEM images (Figure 2B,D). Meanwhile, in the Ouzo domain of HD (WHD = 0.2%, Wpolymer = 0.05%), NCs with diameters ∼350 nm are obtained (DLS, dv/dn = ∼1.10 at Wacetone = 0.69 or 0.66, respectively; Figure 2C,E). It is well-established that positively charged (rather than neutral or negative) carriers are efficiently internalized by cells. For instance, copolymers comprising HPMA and 5 or 20 mol % of 2-(dimethylamino)ethyl methacrylate (DEMA) or 2(methacryloyloxy)ethyl trimethylammonium chloride were shown to favor uptake in C4−2 cells (without inducing cytotoxicity up to 0.4 mg/mL).17 Inspired by these results, we subsequently investigated the preparation of NCs from P(HPMA-co-DEMA) copolymers. Using RAFT copolymerizations, 10, 20, and 30% (molar ratio) of DEMA units were randomly incorporated along PHPMA polymer chains for an overall degree of polymerization again fixed at ∼300 (Table S1). Three precisely defined copolymers, that is, P(HPMA286co-DEMA32), P(HPMA261-co-DEMA83), and P(HPMA223-coDEMA105) were thus obtained (Mn,NMR = 46.2, 50.6, 48.7 kg mol −1 and Đ values below 1.4). Similar to PHPMA homopolymers, ∼80 wt % of acetone is required for nanoprecipitation of P(HPMA-co-DEMA) copolymers. We primarily attempted to construct tertiary amine functionalized NCs with P(HPMA286-co-DEMA32) using a routine “Shift’N’Go” methodology (WHD = 0.05%, WPHPMA = 0.05%, and Wacetone = 0.81). However, no capsule formation was observed under such conditions (Figure 3A). This probably

Figure 3. TEM images of P(HPMA286-co-DEMA32) based nanoobjects at pHs 7 (A), 9 (B), and 10 (C); TEM images of P(HPMA261co-DEMA83) (D) and P(HPMA223-co-DEMA105) based NCs (E); ζpotentials of cationic NCs at pH = 7 (F).

stems from repulsive electrostatic forces, which hamper the adsorption of the chains at the HD droplet surfaces and inhibit the formation of the polymeric shell. The picture was radically different when the “Shift’N’Go” process was performed using basic aqueous solutions of P(HPMA-co-DEMA) copolymers (pH 9 or 10, see Figure 3B−E). As a result of the deprotonation of the copolymers (pKa,PDEMA= 7.3) and the reduction of the repulsive electrostatic forces, polymer chains efficiently aggregated at the interface to form a polymer shell. Noteworthy, at pH 9, a thin, deformable, and fragile polymer membrane was constructed, pleading for inefficient adsorption (presumably because of a partial deprotonation of the amino groups; Figure 3B). A further increase of the pH value to 10 allowed for generating robust NCs with an average diameter of 218 nm (Figure 3C). Similar conditions applied to P(HPMA286-co-DEMA32), P(HPMA261-co-DEMA83), and P(HPMA223-co-DEMA105), resulted in the design of NCs of 449

DOI: 10.1021/acsmacrolett.7b00094 ACS Macro Lett. 2017, 6, 447−451

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ACS Macro Letters comparable size (∼190 nm; Figure 3D,E). ζ-Potential measurements (Figure 3F) were also performed to characterize the surface charge of NCs in aqueous solution (pH = 7). P(HPMA286-co-DEMA32)-, P(HPMA261-co-DEMA83)-, and P(HPMA223-co-DEMA105)-based NCs bear positive charges at the interface, with zeta values of 11.0, 18.3, and 21.3 mV, respectively. We finally prepared tertiary amine and mannose-functionalized PHPMA capsules from a terpolymer obtained by random RAFT copolymerization of EMM, DEMA and HPMA ([HPMA]0/[EMM]0/[DEMA]0 = 7/3/1, P(HPMA230-coEMM102-co-DEMA44), Mn,NMR = 69.7 kg.mol−1 and Đ = 1.31). The cloud-point boundary of this polymer was found at 65% or more of acetone, making both SFME and Ouzo regions of HD accessible for NCs’ construction. Solvent shifting procedures were performed using basic aqueous solutions of P(HPMA230-co-EMM102-co-DEMA44) (pH = 10, WHD = 0.2 wt %, WPHPMA = 0.05 wt %, and Wacetone = 0.66). TEM images show that PHPMA-based NCs possessing around 30% of mannose ligand and 10% of tertiary amine at the surface, and exhibiting rather disparate dimensions, could be straightforwardly obtained in the SFME (dDLS = 90 nm) and the Ouzo (dDLS = 320 nm) regions (Figures 4 and S13). A ζ-potential of

were incorporated into PHPMA chains. Roaming around the phase diagrams and adjusting the pH of the initial aqueous solution, we were able to selectively produce a large series of size-tunable NCs (90−350 nm) with peripheral carbohydrate ligands and positive charges.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00094. Experimental methods, kinetic studies, full characterization of the polymers, and nanocapsules thereof (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Julien Bernard: 0000-0002-9969-1686 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the French Agency for National Research (ANR) for Funding X.Y. postdoc and R.R. PhD (Grant PREPROPOSAL, Reference: ANR-15-CE09-0021).

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REFERENCES

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Figure 4. Route to surface-functionalized PHPMA NCs by the Shift’N’Go process. TEM images of P(HPMA230-co-EMM102-coDEMA44) based NCs. Conditions: pH 10, wHD = 0.2 wt %, wPolymer = 0.05 wt %, acetone mass fraction: 0.66 (A, C) and 0.8 (B).

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DOI: 10.1021/acsmacrolett.7b00094 ACS Macro Lett. 2017, 6, 447−451