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Zwitterionic Polyester-based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time Umberto Capasso Palmiero, Matteo Maraldi, Nicolò Manfredini, and Davide Moscatelli Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00127 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Zwitterionic Polyester-based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time Umberto Capasso Palmiero1,2*, Matteo Maraldi2, Nicolò Manfredini2 and Davide Moscatelli1* 1

Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Via Mancinelli 7 -

20131 Milano, Italy. 2

Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH

Zurich, Switzerland.

KEYWORDS: carboxybetaine, phosphoryl choline, polyester, nanoparticles, ROP, RAFT ABSTRACT: Biodegradable polymer nanoparticles are an important class of materials used in several applications for their unique characteristics. In particular, the ones stabilized by zwitterionic materials are gaining increased interest in medicine as alternative to the more common ones based on poly(ethylenglycol) thanks to their superior stability and ability to avoid both the accelerated blood clearance and allergic reactions. In this work, a novel class of zwitterionic based NPs has been produced and a method to independently control the nanoparticle size, degradation time and polymer molecular weight has been developed and demonstrated. This has been possible by the synthesis and the fine tuning of zwitterionic amphiphilic block-copolymers obtained via the combination of ring opening polymerization and reversible addition-fragmentation chain transfer polymerization. The final results showed that when two block copolymers contain the same number of caprolactone units, the ones with longer oligoester lateral chains degrade faster. This phenomenon is in sharp contrast with the one seen so far for the common linear polyester systems where longer chains result in longer degradation times and it can be used to better tailor the degradation behavior of the nanoparticles.

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INTRODUCTION Polymer nanoparticles (NPs) are an important class of aqueous colloids that find several applications in the biomedical field ranging from the formulation of lipophilic drugs in cancer treatments to the delivery of oligonucleotides in gene therapy1-3. Among all the NPs developed so far, the ones that are structurally composed or stabilized by biodegradable amphiphilic polymers, such as polyester-based block-copolymers, have the advantage to degrade into biocompatible and easily removable compounds and to avoid the use of toxic surfactants4-6. While the lipophilic portion usually consists of biodegradable polyesters and serves as the forming unit of the NP core, the second block of the copolymer usually consist of a hydrophilic polymer that is responsible for the NP stabilization as long as it locates on the NP surface. In this latter case, polyethylen glycol (PEG) has been considered the golden standard due to its ability to reduce NP opsonization by the immune system and to prolong the residence time into the body7, 8. However, recently literature showed that PEG induces the production of specific immunoglobulin M (IgM) antibodies9, 10. These antibodies stimulate the complement system leading to a faster clearance of the PEGylated therapeutics in their subsequent administrations (the so called “accelerated blood clearance effect”)11-14. This side effect may render completely ineffective therapies based on PEGylated NPs, especially when repeated doses are necessary15-17. In addition, previous studies have shown an already high occurrence of these antibodies in 25% of the healthy blood donors probably because of the large use of this hydrophilic polymer as additive in consumer products and in commercially available drug formulations for topical and parental administration15,

18

. This pre-existing

immunization can lead also to first-exposure allergic reactions. In a recent phase 2b clinical trial of a PEGylated RNA aptamer, the occurrence of three serious allergic reactions (one of the which lifethreating) in patients with acute coronary syndrome led to the early termination of this study19. For this reason, alternative solutions have been investigated and novel promising polymers that do not show accelerated blood clearance and allergy reactions, such a poly(n-(2-hydroxypropyl) 2 ACS Paragon Plus Environment

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methacrylamide) (poly-HPMA)20, particular,

poly(carboxybetaine

21

and some zwitterionic polymers have been found22, methacrylate)

(poly-CB)

and

23

. In

poly-(phosphorylcholine

methacrylate) (poly-MPC) have shown not only the ability to avoid protein adsorption, polymerspecific antibody production and to stabilize the NPs, but their high hydrophilicity allows also to reduce or avoid cryoprotectants in the lyophilization step that is generally required for the NP storage24-26. However, their low solubility in the common organic solvents makes difficult to produce libraries of amphiphilic copolymers based on these zwitterionic monomers and to control and tune the properties of the final material26, 27. In the last years, the so called “macromonomer method” have found increasing attention as an easy way to produce oligoesters that bear a double bond and, in turn, that can be used to produce biodegradable NPs via free radical polymerization (FRP)28. This procedure consists in the synthesis of a macromolecular monomer via the ring opening polymerization (ROP) of a lactone, such as caprolactone, in the presence of a monomer that bears a hydroxyl group. The possibility to produce a “brush like” polymer via FRP have been found to be an effective way to obtain NP without the use of long polyester chains that are usually difficult to be solubilized in many solvents. In addition, this particular architecture allows an additional degree of freedom into the final design of the material by varying the length of these brushes; this can be used for example to control the NP degradation time29. However, the use of the FRP does not allow the control over the NP size and of the molecular weights of these copolymers. The advent of the controlled living polymerizations, such as reversible additionfragmentation chain transfer (RAFT) polymerization30, has paved the way to the control over the molecular weight and structure of the polymers that are made via radical polymerization. It has opened the possibility to produce well-defined block-copolymer and to study their behavior once organized into NPs. In particular, a simplified geometrical model able to correlate the NP size with the length of the hydrophilic and lipophilic part of the forming block copolymer has been introduced and a method to decouple these important parameters have been found via the adoption of macromolecular PEG surfmers as hydrophilic portion of the block copolymers31. However, in 3 ACS Paragon Plus Environment


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this case, the NPs are obtained via ab initio emulsion polymerization of methyl methacrylate and are not biodegradable. Moreover, they are stabilized by a polymer (PEG) that can cause allergic reactions as above-mentioned. In this work, a method to independently control the main characteristics of biodegradable NPs stabilized by zwitterionic polymers is presented. The method consists in the synthesis of a brush-like structure via the combination of two living polymerization: RAFT and ROP. In this way, in contrast to what happen with the common biodegradable linear block-copolymers, it is possible to not only control the length of the two parts of the block copolymers (n, p, Figure 1), but also the length of the brushes (q, Figure 1). This permits to decouple the NP size and the block-copolymer molecular weight, and to obtain NPs with the same number of caprolactone units, but with different degradation time. In order to demonstrate this possibility, a library of block copolymers based on zwitterionic polymers (i.e poly(CB) and poly(MPC)) have been produced and self-assembled into water to generate NPs with different size and block-copolymer molecular weights. As long as these NPs are intended for biomedical applications, the degradation behavior of these colloids has been studied and correlated with the structure of the lipophilic part of the block copolymer. The final results showed that when two block copolymers contain the same number of caprolactone units, the ones with longer oligoester lateral chains degrade faster.

MATERIALS AND METHODS Materials. ε-Caprolactone (CL, 97% MW= 114.14), stannous octoate ([Sn(Oct)ଶ ], MW=405.12),

2-hydroxyethyl

methacrylate

(HEMA,

97%,

MW=

130.14),

4-cyano-4-

(phenylcarbonothioylthio)-pentanoic acid (CPA, 97%, MW=279.38), 4-4’azobis (cyanovaleric acid) (ACVA, 98%, MW= 280.2), 2-Methacryloyloxyethyl phosphorylcholine (MPC, 97%, MW= 295.27), 2-(dimethylamino)ethyl methacrylate (DAEMA, 98%, MW= 157.21), tert-butyl


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bromoacetate (98%, MW= 195.05), ethanol (EtOH, 99,8%) were purchased from Sigma Aldrich and used as received. All the solvents and the chemicals used were analytical-grade purity and were purchased from Sigma-Aldrich. Synthesis of PCL-based Macromonomer. Three different oligoester bearing monomers (HEMA-CLq) were synthesized via ROP of CL in bulk with HEMA as co-catalyst and SnOct2 as catalyst according to a previous work28. The HEMA to SnOct2 ratio was set equal to 1/200 while the monomer to initiator molar ratio (q) was set equal to 3, 5 and 7. As an example for HEMA-CL5, 17.54 g of CL and 175 mg of Na2SO4 were weighted in a septa-sealed flask and heated to 130°C in a constant temperature oil bath under stirring. 4 g of HEMA were mixed with 62 mg of tin octanoate in a different vial and injected into the pre-heated CL containing flask. After 3 h, the reaction was stopped and an aliquot was taken to perform

1

H-NMR (MeOD) and GPC.

Caprolactone conversion (X) and the average number of caprolactone units attached to HEMA (qNMR) were evaluated via 1H-NMR according to a previous protocol28 and reported in Table S1 in the Supporting Information. Macromonomer number-averaged molecular weight (MnGPC) and dispersity (ÐGPC) (Table S1 in Supporting Information, SI) were evaluated via gel permeation chromatography (GPC) with a Jasco Series Apparatus. For GPC analysis, the samples were dissolved at 0.4 mg/ mL in THF and filtered through a 0.45 µm pore-size membrane before injection. The separation was performed at 35°C and at a flow rate of 0.5 mL/min. MnGPC and ÐGPC were determined from differential refractive index (RI) data and were relative to poly(styrene) standards (from 580 to 197 300 g/mol). Synthesis of the Precursor of CB Monomer. A protected CB monomer was synthesized via addition of tert-butyl bromoacetate with DAEMA as already reported in literature26. Briefly, 11 ml of DAEMA and 19 ml of tert-butyl bromoacetate were dissolved in 60 ml of acetonitrile in a 5 ACS Paragon Plus Environment


round bottom flask with a magnetic stirrer. The mixture was left to react at 50°C under a protective atmosphere of nitrogen for 2 days and then evaporated under vacuum. The final product was washed three times with diethyl ether, dried under vacuum and characterized via 1H-NMR (MeOD). Synthesis of the Zwitterionic Hydrophilic Blocks. Two zwitterionic hydrophilic blocks were synthesized via RAFT polymerization of CB or MPC in a mixture of 50/50 v/v ethanol/3 mM acetic buffer in the presence of CPA as RAFT agent and ACVA as initiator. The monomer to CPA molar ratio and the ACVA to CPA molar ratio were set equal to 25 and 1/3, respectively. In the case of the MPC-based hydrophilic block (25MPC), 2.23 g of MPC, 84 mg of CPA and 28mg of ACVA were dissolved in 10 ml of ethanol/acetic buffer mixture and poured in a septa-sealed flask. The solution was purged with nitrogen for 15 min and left to react for 24 h at 65°C under magnetic stirring. The mixture was dialyzed with a SpectraPor® RC membrane (MWCO=3.5kDa) against deionized water with a small quantity of HCl (pH = 3-4) for 1 day to eliminate the buffer salt, the unreacted monomer and at the same time to avoid the hydrolysis of the RAFT agent. The final polymer was precipitated in acetone, recovered as a red solid and stored at -20°C. The choice of the solvent was driven by solubility issues as long as MPC, CB and their corresponding polymers are only soluble in methanol and water while CPA is soluble in ethanol, but not in water. An aqueous medium has been chosen instead of pure methanol due to the higher polymerization rate of hydrophilic monomers in water compared to organic solvents. In addition, the presence of an acidic environment avoids the hydrolysis of the RAFT agent and at the same time allows the direct elimination of the ter-butyl protective group of the CB, as visible in the 1H-NMR spectrum in Figure S1 (SI). An aliquot of the MPC or CB polymerization was withdrawn before and after purification and analyzed via 1H-NMR (10 mg were dissolved in a mixture of 0.8 mL MeOD and 0.3 mL DMSO-d6). Monomer conversion (X) and the degree of polymerization (nNMR) were evaluated from the 1H-NMR spectra (Figure S2-3, SI) according to equation S1-4 (SI) and reported in Table S1. The block-copolymers of the library are identified as nCB-pCLq or nMPC-pCLq 6 ACS Paragon Plus Environment

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according to the structure shown in Figure 1. As an example, 25MPC-80CL5 is the block copolymer composed of 25 units of MPC and 80 lipophilic brushes with 5 units of caprolactone. The n, p and q values reported in the name of the block-copolymers are the theoretical ones while the actual values obtained via 1H-NMR are reported in Table S1 and cited in the main text. Synthesis of the Zwitterionic PCL-based Block-Copolymers. A library of amphiphilic block copolymers was obtained via RAFT polymerization of HEMA-CLq in the presence of the zwitterionic hydrophilic block (25MPC or 25CB) as RAFT agent and ACVA as initiator. The ACVA to 25MPC or 25CB molar ratio was set equal to 1/3 and the HEMA-CLq (q = 3, 5, 7) to 25MPC or 25CB molar ratio (p) was varied from 5 to 80 in order to obtain 18 polymeric surfactants for each zwitterionic hydrophilic block. As an example, for the 25MPC-20CL5, 0.209 g of 25MPC, 0.384 g of HEMA-CL5 and 2.55 mg of ACVA were dissolved in 3 ml of methanol and poured in a 4 ml vial. The mixture was purged with nitrogen for 2 min and left to react at 65°C under stirring for 24 h. Then the copolymer was precipitated in diethyl ether several times, dried under vacuum and stored at -20°C. In the case of the block-copolymers synthesized with HEMA-CL7, a mixture of methanol/DMSO (50/50 v/v) was used as reaction solvent due to the high lipophilicity of the macromonomer and of the resulting product. An aliquot of the mixture was analyzed before and after the purification via 1H-NMR (10 mg were dissolved in a mixture of 0.8 mL MeOD and 0.3 mL DMSO-d6). Macromonomer conversion (X) and the degree of polymerization (pNMR) were evaluated from the 1H-NMR spectra (Figure S4-5, SI) according to equations S5-8 (SI) and reported in Table S2-3. NP Synthesis. NPs were synthesized via self-assembly of the zwitterionic PCL-based block copolymer. Briefly, 10 mg of the copolymer was dissolved in 200 µl of a methanol/DMSO mixture (75/25 v/v) and then added dropwise into 3 ml of PBS under strong stirring. Different methanol/DMSO mixtures (0/100, 25/75, 50/50, 75/25 and 100/0 v/v) were adopted in order to study the effect of the organic solvent composition on the final NP size (Dn). All the NPs were 7 ACS Paragon Plus Environment


synthesized three times. NP size (Dn), polydispersity (PdI), and ζ-potential were evaluated via dynamic light scattering (DLS) on a Zetasizer (Malvern, Instruments). The NPs were diluted 10 times with deionized water before DLS measurement. All the results reported are an average of three measurements and Dn values are relative to volume (Table S2-5, SI). Scanning electron microscope (SEM) was performed on a Zeiss Leo 1530 operated at an accelerating voltage of 3kV. The latex was coated in a Safematic CCU-010 HV using a Pt-Pd (80-20) target, whereas the layer thickness was set at 4 nm. NP Degradation Test. The degradation kinetic of the NPs was investigated at different pH at 37°C via DLS by acquiring the intensity of the scattered light (count rate) and NP average size modifying a previously published protocol32, 33. Briefly, 1.5 ml of the NPs were mixed without further purification with 1 ml of 0.1 M HCl (pH = 1), 3 mM acetic buffer (pH = 5.4), 0.1 M phosphate buffer (pH = 8), or 0.1 M NaOH (pH = 14). The half-life (t1/2) of each NP was evaluated as the time at which the intensity of the scattered light reaches half of the initial value. This parameter was obtained via interpolation of the experimental data. All the degradation tests were repeated three times. RESULTS AND DISCUSSION Block Copolymers Synthesis. Two libraries of block copolymers were synthesized via a three steps procedure that consists in a ROP and two subsequent RAFT polymerizations. In the first step, three PCL-based macromonomers were synthesized via ROP of CL in the presence of HEMA as initiator in order to produce oligo-esters chains with three different lengths (q = 3, 5, 7) that are able to polymerize via free radical chemistry. In the second step a CB or MPC hydrophilic block was synthesized via RAFT polymerization of the corresponding monomer in an acetic buffer/ethanol mixture. The last step consisted in the RAFT polymerization of the HEMA-CLq in the presence of the previously synthesized zwitterionic hydrophilic block and it was performed in 8 ACS Paragon Plus Environment

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methanol or methanol/DMSO mixture. The high hydrophilicity of the zwitterionic polymers makes the last step of block-copolymer synthesis critical because of the high difference in lipophilicity between the two blocks. In fact it is difficult to find solvent able to dissolve both the parts of the block copolymers as also reported in literature for the synthesis of linear block-copolymers obtained via direct conjugation of a polyCB with a PLGA chain26. However, in this work, the choice of a comb-like structure of the final block-copolymer with biodegradable side chains that bears a hydroxyl group allows a higher solubility in the reaction mixture, a higher tunability of the lipophilicity and, thus, allows to produce a large library of block-copolymers (Table S2-S3, SI). In addition, the choice of a controlled polymerization permits to finely tune the number of the zwitterionic monomers in the hydrophilic portion (n) and the length (q) and the number (p) of the oligoester side chains of the lipophilic part, as visible in Figure 1.

Figure 1. General structure of a comb-like polymer obtained via the macromonomer method where Rh represents a generic hydrophilic group, such as phosphoryl choline (MPC) or carboxybetaine (CB).



NP Synthesis. Due to the amphiphilic properties of the block-copolymers, the synthesized materials can directly self-assemble into NPs if dissolved in an organic solvent and then added dropwise to an aqueous medium, such as PBS. In particular, in an ideal geometrical disposition of block-copolymers that forms the NP, the lipophilic part of the polymeric surfactant is segregated in the core of the NP while the hydrophilic portion is confined on the NP surface and provide steric stabilization to the colloid34, as depicted in Figure 2. In the case of the zwitterionic blockcopolymers, the surface is expected to be composed of an equal number of positive and negative charges leading to a ζ-potential close to 0. However, a slightly negative value is obtained via DLS measurement for both the NPs stabilized by polyMPC (ζ-potential = - 10 ± 7 mV for 25MPC60CL5 at pH = 7.4) and polyCB (ζ-potential = - 11 ± 9 mV for 25CB-60CL5 at pH = 7.4) due to the presence of the carboxylic group at the end of the block copolymer (Figure 2). However, when the solution is adjusted with 0.1 HCl to pH = 1, the ζ-potential values turns positive due to the protonation of the phosphate group of the phosphoryl choline (ζ-potential = 11 ± 8 mV for 25MPC60CL5 at pH = 1) and of the carboxylic group of the carboxy betaine (ζ-potential = 40 mV ± 8 mV for 25CB-60CL5 at pH = 1), in agreement with what already shown in literature35, 36.


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Figure 2. Synthesis of the PCL-based block copolymers stabilized by polyMPC and their selfassembly behavior. In the case of amphiphilic species obtained via RAFT polymerization, it is possible to correlate the NP Dn with the monomer units of the lipophilic part (p in this work) according to eq. (1)31:

‫= ݊ܦ‬

଺·௣·ெௐಹಶಾಲష಴ಽ೜

(1)

ேಲೡ೚ ·஺಴೚ೡ ·ఘ್_೗೔೛೚

where MWHEMA-CLq is the molecular weight of the lipophilic monomer, ρb_lipo the density of the lipophilic part of the block copolymer, Navo the Avogadro number, and ACov the surface area that the hydrophilic part of the block copolymer can cover. This equation has been derived from the hypothesis that the NP size depends on the number of the amphiphiles and on the volume that their lipophilic portion is able to occupy, a behavior well-documented in literature34,

37

(we leave the

detailed demonstration and explanation of eq.1 to the ref31). As visible in Figure 3a, Dn is a linear function of p for the CB and MPC-based block-copolymers synthesized with q = 5.3, as predicted from eq.1. This linear behavior is consistent with what already shown in literature for other NPs composed of block-copolymers and obtained via heterogenous polymerizations (e.g emulsion or dispersion polymerization)31,

38-41

. In addition, all the NPs synthesized in this work present a

spherical shape, as visible from the SEM image of the NPs composed of a MPC-based block copolymer (Figure 3b). Interestingly, the MPC-based NPs have a smaller size compared to CBbased ones at the same p and q indicating that the MPC block may be less prone to be buried inside the NP core due to its higher hydrophilicity. In fact, it is worth to mention that, as long as the NP PdI are in general higher (Table S2-5, SI) compared to NPs obtained via other techniques, such as emulsion polymerization31, 42, due to the difficulties in the chain rearrangement during a common nanoprecipitation step, a perfectly homogenous and ordered configuration of the block-copolymers in all the NPs is very unlikely. On the contrary of micelles produced using surfactants, nanoprecipitation generates kinetically frozen NPs that are not in a thermodynamic equilibrium due 11 ACS Paragon Plus Environment


to diffusion limitations43. If the block-copolymers have not enough time to rearrange, some of the hydrophilic chains may be buried inside the core affecting their ability to stabilize the NP and, thus, resulting in a lower ACov and bigger size44. For this reason, the choice of the organic solvent necessary to dissolve the amphiphilic block-copolymers plays an important role on the characteristics of the final NPs as long as its diffusion and affinity with water impact the selfassembly of these colloids. While methanol and water are the only two known solvents able to solubilize these zwitterionic polymers26, the lipophilic brush-like portion can be dissolved in several water soluble organic solvents (e.g. methanol, ethanol, DMF, THF and DMSO), but obviously not in water. For this reason, methanol has been adopted as the main component of the organic phase. However, as long as the lipophilic portion becomes insoluble in methanol at high molecular weights, a mixture with different concentrations of DMSO (ranging from 0 to 100 vol.%), a low cytotoxic compound45-47, has been adopted in the nanoprecipitation step in order to study the effect of the solvent composition on the NP size, as shown in Figure 3 c,d.


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9 9 . 0 = R

2

a 160

Dn (nm)

9 9 . 0 = R

2

80

MPC CB Linear Fit 0 0

40

80

p (-)

270

300

c

25MPC-5CL3 25MPC-40CL5 25MPC-80CL7

240 210

d

25CB-5CL3 25CB-40CL5 25CB-80CL7

270 240 210

180

180 150

Dn (nm)

Dn (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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120 90

150 120 90

60

60

30

30

0

0 0

50

100

0

CDMSO (vol.%)

50

100

CDMSO (vol.%)

Figure 3. NP Size as a function of p for CB and MPC block-copolymers with q = 5.3 (a). SEM image of NPs composed of the MPC-block copolymer with q = 5.3 and p = 84 (b). NP Size as a function of the organic solvent composition for MPC (c) and CB (d). In the case of zwitterionic polymer surfactants with small lipophilic portions (25MPC-5CL3 and 25CB-5CL3), the same NP Dn can be obtained across a wide range of CDMSO, but not with pure DMSO where the high content of zwitterionic moieties makes this block-copolymers insoluble. In the case of the highest p and q synthesized in this library (25MPC-80CL7 and 25CB-80CL7), the DMSO has a beneficial effect on the NP synthesis reducing the final size indicating that this mixture is able to better dissolve the block-copolymer, but at CDMSO = 100 vol.% the short hydrophilic portion becomes insoluble and probably generates inverse micelles. These blockcopolymers, however, are not soluble in pure methanol and it is not possible to obtain NPs using the pure solvent. The block-copolymer with intermediate p and q, such as 25MPC-40CL5 and 25CB13 ACS Paragon Plus Environment


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40CL5, present a reduction or increase in the NP size at increased CDMSO depending on the lipophilic/hydrophilic balance of the block-copolymers. It is noteworthy to mention that the solvent composition can be used as another parameter for the fine tuning of the final NP size as long as all the NPs here produced do not present a significant change in PdI (Table S3-4) due to the fact that they are in any case in a kinetic frozen state44. More conveniently, this additional degree of freedom of the system can be exploited to increase the loading of therapeutics with different polarity and, thus, different solubility when they are directly incapsulated into NPs during the nanoprecipitation step33. In general, the mixtures with CDMSO = 25, 50 and 75 vol.% seem to be all suitable for the production of a library within the values of p and q here adopted. For this reason, CDMSO = 25 vol.% has been chosen as an example. In an ideal geometrical configuration of the block-copolymers synthesized in this work, the entire volume of the NP core is composed by the lateral oligo-ester chains and the backbone of the lipophilic block. This particular feature can be used to decouple the NP size and the copolymer molecular weight in a similar manner to what already shown in literature by modifying the ability of the hydrophilic block to stabilized the NPs (Acov)31. In fact, as long as the lipophilic monomer is obtained via ROP, MWHEMA-CLq can be expressed as28:

‫ܹܯ‬ுாெ஺ି஼௅௤ = ‫ܹܯ‬ுாெ஺ + ‫ܹܯ ∗ ݍ‬஼௅

(2)

where MWHEMA and MWCL are the molecular weight of HEMA and CL, respectively. If we combine eq.(1) with eq.(2), it is possible to obtain the following correlation:

‫= ݊ܦ‬

଺·௣·(ெௐಹಶಾಲ ା௤∗ ெௐ಴ಽ )

(3)

ேಲೡ೚ ·஺಴೚ೡ ·ఘ್_೗೔೛೚

where the length (q) and the number (p) of the lipophilic brushes can be independently chosen at a given NP Dn. This is experimentally in agreement with what visible in Figure 4a where the NP Dn produced via the self-assembling of poly(MPC)-based block copolymers with q = 3.2, 5.3, 7.7 are 14 ACS Paragon Plus Environment

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reported as a function of p. Interestingly, also in the case of q = 3.2 and q = 7.7 it is possible to see a linear behavior and that the slopes of the linear trend for q = 3.2 and q = 7.7 are lower and higher than the one with q = 5.3, respectively, in good agreement with what expected from eq.(3). The results are less clear for the poly(CB)-based block-copolymers where it is possible to obtain higher NPs with q = 7.7 compared to the block copolymers with q = 3.2 and 5.3 at low p (Figure 4b). On the contrary, they present a high Dn at high p probably due to the organic solvent effect mentioned above. However, NPs with a Dn ranging from 20 to 140 nm with poly(MPC)-based block copolymers and from 30 to 200 nm for poly(CB)-based ones can be easily obtained by finely tuning the p and q parameters.

a

b

240

4 9 . 0 = R

2

160

Dn (nm)

7 9 . 0 = R

Dn (nm)

9 9 . 0 = R

2

160

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

80

80

q = 3.2 q = 5.3 q = 7.7

q = 3.2 q = 5.3 q = 7.7

Linear Fit 0

0

0

40

80

0

p (-)

40

80

p (-)

Figure 4. NP Size as a function of p for poly(MPC) (a) and poly(CB) (b) based block-copolymers at three different q.

It is important to note that the possibility to finely tune the NP Dn and the block-copolymer molecular weight in a system intended for biomedical applications is of paramount importance as long as it is well known that the NP Dn affects the therapeutics’ distribution when injected intravenously and that the block-copolymer molecular weight influences the NP accumulation into the body48, 49.



NP Degradation Behavior. The degradation behavior of the NPs obtained via the selfassembly of poly(MPC)-based block-copolymers was evaluated at 37°C via DLS measurements by collecting the intensity of the scattering light and the NP size at different time points and at different pH (Figure S6). As long as the degradation behavior is faster at higher pH as visible from the Figure S6 and also well-documented in literature50, we have chosen to use a pH = 14 to take a quick look at the relative behavior of the NPs in a similar way to what already done in other degradation studies of polycaprolactone51,

52

. The evolution of the relative scattering intensity

(normalized to the intensity at the time t=0) and of the NP size is reported for poly(MPC)-based NPs with three different p for each q (Figure 5a-f). In the case of the NPs obtained with q = 3.2, the degradation kinetic is faster as the p decreases (p from 80 to 40) (Figure 5a). A similar NP degradation kinetics can be seen also in the case of block-copolymers that present a longer lipophilic lateral chain (q = 5.3 and 7.7 in Figure 5c,e). The increase of the length of the lipophilic portion of the block-copolymer that compose the NP slow down the degradation kinetic independently from the length of the single brush. This behavior is reasonable if we consider that more ester breakages at higher p are necessary to make the block-copolymer completely soluble in water. At the same time, the NP Dn does not change significantly during time independently from the structure of the lipophilic part of the block-copolymer (Figure 5b,d,f). This latter phenomenon indicates that the breakage of the ester bonds during the degradation does not lead to a remarkable swelling of the NP as shown in other systems33. The disassembly of the block-copolymers and, in turn, the disappearance of the NP likely depends on the reduction to a critical limit of the lipophilicity of the poly-caprolactone based part of this amphiphilic species. This critical lipophilicity value is expected to be dependent on the hydrophilic/lipophilic balance (HLB) of the block-copolymer and to be an intrinsic characteristic of the material. In fact, it is very unlikely that the disappearance of the NPs coincides with the complete degradation of the oligo-ester chains by a sequential breakage of the ester units. It is more probable that the ester breakage occurs randomly along the lipophilic lateral chain leading to the production of shorter oligoester species of length i 16 ACS Paragon Plus Environment

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and a less lipophilic portion of the block-copolymer, as depicted in Figure 6a. Only at the end of the degradation process, a completely hydrophilic poly(HEMA) backbone and p·q number of caprolactone units (nCL) are expected to be released. In fact, it is well known that poly-caprolactone polymers present a bulk-type degradation that starts after complete diffusion of the water inside the polymeric matrix, in sharp contrast to what happen with polymers, such as polyanhydrides, that presents an erosion-type degradation mechanism53-55.


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As long as it is possible to control the number and the length of the lipophilic brushes, and, in turn, the disposition of the points of breakage, the half-life (t1/2) of the NPs have been evaluated and characterized against the overall amount of caprolactone units of the lipophilic block (nCL = q·p) at given q (Figure 6b) to study the impact of the different block-copolymer structure on the degradation behavior of the NPs. The increase of the overall nCL lead to an increase in t1/2 independently upon the disposition of the caprolactone units. This is consistent with what already seen in Figure 5a,c,e and indicates that the higher the number of point of breakages, the higher the time necessary for the block-copolymers to reach the critical limit of disassembly. A similar result have also been found in the degradation behavior of the NPs obtained via the FRP of similar oligoester-based macromonomers29. However, in this latter case, as long as the oligoester lateral chains are incorporated in a random copolymer structure with PEG lateral chains and that the number of biodegradable lateral chains is difficult to estimate due to the non-controlled nature of the process, it is not possible to study and, thus, to evaluate the single impact of the q and p parameters on the t1/2. On the contrary, in this case, the RAFT polymerization allows to finely control p. Interestingly, a lower number of caprolactone units in the single lateral chain leads to a higher increase of t1/2, as visible from the slopes of the linear fits in Figure 6b. The disappearance of the NPs is faster when the lipophilic part of the block-copolymer has a higher q at the same number of caprolactone units indicating that the disposition of the points of breakage is an important factor that can be used to tune the degradation behavior of the NPs. In fact, as long as the oligoester chains with i from 1 to q that derived from the degradation of the lateral chains (Figure 6a) present already a not negligible solubility in water due to their relatively low molecular weight, the NPs with higher q requires less breakages to be dissolved compared to the ones with lower q and higher p.


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Figure 6. General mechanism of degradation of the lipophilic part of the block-copolymer. (a) Half-life of the NPs evaluated as a function of the overall number of caprolactone units (nCL) that the lipophilic part of the block-copolymer contains. (b) nCL is evaluated as nCL= p·q. This behavior is in sharp contrast to what shown in literature for the NPs obtained via the selfassembly of polyester-based linear block-copolymers where higher caprolactone units in the lipophilic chain results in bigger NPs and longer degradation time55, 56. In this case, the degradation 20 ACS Paragon Plus Environment

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time and the NPs are directly correlated with the molecular weight of the block-copolymers resulting in lower tunability and the impossibility to decouple these parameters56. On the contrary, the NPs that present brush-like structure in the lipophilic portion of the block-copolymers can be finely tuned paving the way to an independent selection of the degradation time and NP size. In fact, with this method it is possible to produce NPs with the same size, but with different degradation times or vice versa depending on the requirement of the biomedical application.

CONCLUSIONS In this work, a class of zwitterionic NPs has been produced via the self-assembly of brushlike amphiphilic block-copolymers. The synthesis of these polymeric surfactants via the macromonomer method and its combination with RAFT polymerization have allowed the fine tuning of their structure and, in turn, to independently control the NP size and molecular weights of the forming copolymer. The effect of the solvent composition in the nanoprecipitation step has been studied and found an important parameter that influences the NP size. In the end, the degradation behavior of these NPs has been studied and correlated to the number of caprolactone units present in the block-copolymers and to their architectural disposition (p, q). It has been found that NPs with the same number of caprolactone units, but with longer lipophilic brushes, degrade faster in sharp contrast to what happen for the common linear polyester-based block-copolymers synthesized so far. This brush-like architecture and the method here developed can be used to finely and independently tailor the NP size and degradation behavior of these zwitterionic-based materials paving the way to advanced applications in biomedical field.

ASSOCIATED CONTENT Supporting Information The supporting information are available free of charge on the ACS publication website.



1

H-NMR spectra and characterization of the zwitterionic hydrophilic blocks, and block-

copolymers; Evolution of the relative scattering intensity for the NPs at different pH; Characterization of HEMA-CLq, nCB and nMPC; MPC or CB-based block-copolymers and NP characterization; NP Size and PdI of the NPs composed of CB or MPC-based block copolymers at different DMSO content.

AUTHOR INFORMATION Corresponding Authors *

e-mail: [email protected].

*

e-mail: [email protected].

ORCID Umberto Capasso Palmiero: 0000-0002-9683-491X Davide Moscatelli: 0000-0003-2759-9781 Notes The authors declare no competing financial interest

ACKNOWLEDGMENT We thank Antoine Klaue (ETH) for SEM images.

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For Table of Content Use Only Zwitterionic Polyester-based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time Umberto Capasso Palmiero1,2*, Matteo Maraldi2, Nicolò Manfredini2 and Davide Moscatelli1* 1

Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Via Mancinelli 7 -

20131 Milano, Italy. 2

Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH

Zurich, Switzerland.


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