Amphiphilic and Hydrophilic Block Copolymers from Aliphatic N

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Amphiphilic and Hydrophilic Block Copolymers from Aliphatic N-Substituted 8 Membered Cyclic Carbonates: A Versatile Macromolecular Platform for Biomedical Applications Shrinivas Venkataraman, Jeremy PK Tan, Victor W. L. Ng, Eddy W.P. Tan, James L Hedrick, and Yi Yan Yang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01463 • Publication Date (Web): 20 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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Amphiphilic and Hydrophilic Block Copolymers from Aliphatic N-Substituted 8-Membered Cyclic Carbonates: A Versatile Macromolecular Platform for Biomedical Applications

Shrinivas Venkataramana*, Jeremy P. K. Tana, Victor W. L. Nga, Eddy W. P. Tana, James L. Hedrickb* and Yi Yan Yanga*

a

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore

138669, Singapore

b

IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA

*

Corresponding authors:

Tel: 65-6824-7106; Fax: 65-6478-9084; E-mail: [email protected] Tel: 65-6824-7192; Fax: 65-6478-9084; E-mail: [email protected] Tel: 1-408-927-1632; Fax: 1-408-927-3310; E-mail: [email protected]

Keywords Block copolymers, polycarbonates, pH-responsive, cationic polymers, zwitterionic polymers, drug delivery applications

ACS Paragon Plus Environment

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Abstract Introduction of hydrophilic components, particularly amines and zwitterions onto a degradable polymer platform, while maintaining precise control over the polymer composition, has been a challenge. Recognizing the importance of these hydrophilic residues in multiple aspects of the nano-biomedicine field, herein, a straight-forward synthetic route to access well-defined amphiphilic and hydrophilic degradable block copolymers from diethanolamine-derived functional eight-membered N-substituted aliphatic cyclic carbonates is reported. By this route tertiary amine, secondary amine and zwitterion - residues can be incorporated across the polymer backbone. Demonstration of pH-responsiveness of these hydrophilic residues and their utility in the development of drug-delivery vehicles, catered for the specific requirements of respective model drugs (doxorubicin and diclofenac sodium salt) are shown. As hydrophilic components in degradable polymers play crucial roles in the biological interactions, these materials offers opportunities to expand the scope and applicability of aliphatic cyclic carbonates. Our approach to these functional polycarbonates will expand the range of biocompatible and biodegradable synthetic materials available for nano-biomedicine, including drug- and gene-delivery, antimicrobials and hydrophilic polymers as poly(ethylene glycol) (PEG) alternatives.


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Introduction Block copolymers, two or more types of polymers (with different chemical structure and properties) attached via covalent bond, constitute an important class of materials. The solution properties of block copolymers with the selective solvation of one block over the other can generate supramolecular assemblies such as micelles. In the biomedical milieu, self-assembled block copolymers have found numerous applications.1-3 For example, hydrophobic drugs can be partitioned into the core of a self-assembled amphiphilic block copolymer, rendering the drug dispersible in aqueous environment for increased bioavailability and improved therapeutic efficacy4, and proteins can be selectively encapsulated in a double-hydrophilic diblock copolymer and released under a specific biochemical trigger.5-6 These strategies offer a simple and versatile approach to deliver therapeutics in an efficient manner that is otherwise challenging. Moreover, the interfacial curvature of the ensembles of block copolymers can be tailored by controlling the relative block ratios, leading to programmable self-assemblies.7 As the physicochemical attributes of the self-assembled soft nanostructures have been shown to significantly impact the interactions with the physiological environments,8-9 the ability to tailor the assemblies using controlled polymerization strategies and orthogonal chemistries10 offers a tool-box for custom designing materials. For the biomedical applications, it is highly desirable to construct these nanostructures from materials that are biocompatible and biodegradable.11

Efficient synthetic access to functional degradable polymers with tailored compositions from inexpensive and commercially available starting materials will impact not only high-end biomedical applications, but also consumer-care and packaging applications. Tremendous progress has been made in the development of potent catalysts12-13 and synthetic routes to access


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functional cyclic monomers amenable for ring-opening polymerization (ROP)14 to produce various classes of degradable materials, suitable for biomedical applications.15-18 Among degradable materials, aliphatic polycarbonates have emerged as an important class of polymers, primarily due to the unprecedented ease in introduction of functional groups at both monomer1932

and post-polymerization stages.21-25, 33-39 Novel step-efficient synthetic strategies have also

been developed to access a variety of well-defined functional polycarbonates from inexpensive starting materials.40-44 Recently we reported a step-efficient approach to access N-substituted 8membered aliphatic cyclic carbonates from diethanolamine and other commercially available chemicals.42 Based on the N-substituent, different sub-classes of monomers were synthesized (Naryl, N-alkyl and N-carbamate). These 8-membered carbonates can be well polymerized by an organocatalytic ROP route to generate narrowly dispersed polymers of predictable molecular weights and the polymerization kinetics of these monomers was found to be highly dependent on the catalyst and N-substituent. One of the key distinguishing features of these polymers obtained from aliphatic N-substitued 8-membered cyclic carbonates is the ability to readily introduce functionalities such as secondary and tertiary amines along the polymer back-bone, which is otherwise challenging.35 Ability to impart hydrophilicity through introduction of chemical functionalities like hydroxyl, amine or carboxylic acid groups would enable control over properties such as surface wettability, rate of degradation, biocompatibility and sequestration of cargo, and could also serve as reactive handles for subsequent transformations to append ligands, impart biological functions (like antimicrobial properties, cell adhesion, targeting specific population of cells).11 For example, installation of tertiary amines across the polymer backbone via polymerization of 8-membered carbonates, followed by quaternization resulted in broad spectrum antimicrobials.45


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Incorporation of cationic residues onto biodegradable polycarbonates is also desirable for development of drug delivery carriers. Moreover, cationic components impart pH-responsiveness and serve as reactive handles for additional chemistries. Mostly quaternary ammonium salts and tertiary amines are introduced to aliphatic polycarbonates along side-chains via an alkyl halide residue.36,

46-47

In comparison to the functional groups along the side-chains, introduction of

functionalities along the main-chain would lead to differences in physico-chemical properties. To date, direct approach to introduce, these hydrophilic functionalities along main-chain of polycarbonates, is limited.42, 45, 48 In this study, we demonstrate the utility of N-substituted 8membered aliphatic cyclic carbonates in accessing block copolymers incorporating pHresponsive hydrophilic components, across the main-chain of the polymers. By this approach, functionalities such as tertiary amines can be incorporated directly, without any requirement for post-polymerization derivatization steps. Likewise, secondary amines, and zwitterions, can be introduced via simple post-polymeriaztion acidolysis step. So far, there are only limited reports35, 48

on introduction of these functionalities onto aliphatic polycarbonates. It is important to note

that compared to earlier reports, our current approach is not just restricted to tertiary amines,45, 4849

but it does not require relatively expensive reagents too.35 Apart from novelty in terms of

introducing unprecedented hydrophilic functionalities across the main-chain, our motivation to synthesize these functional block copolymers also stems from their potential applicability in drug delivery. Incorporation of amines would impart pH-responsiveness and enhance drug loading capacity50 (for drugs with anionic residues), and the zwitterionic residues would confer antifouling51 properties. The ability to tailor the physico-chemical environment within the nanostructured micelles through the design of block copolymers to suit the requirement of a


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specific drug is important for the successful encapsulation and delivery of therapeutics.52-53 The utility of these functional polycarbonates and also the synthetic versatility to tailor polymer composition for specific drug delivery applications are highlighted with model drugs such as doxorubicin (Dox) and diclofenac sodium salt (DS).


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Experimental section Materials All reagents were purchased from Sigma-Aldrich or TCI or Merck and unless otherwise specified, were used as received. All other solvents used were of analytical grade and used as received unless specifically mentioned. Synthesis of cyclic carbonate monomers used in this study were prepared as described elsewhere.42 Benzyl alcohol and 1,8-diazabicyclo[5.4.0]undec7-ene (DBU) were distilled twice from CaH2 under dry N2 and stored in the glove box. The macroinitiator, mPEG-OH (~ 5.0 kDa, lot # 1211.265, PDI = 1.02) was purchased from RAAP Polymere GmbH (Tuebingen, Germany). Poly(ethylene glycol) macroinitiator, HO-PEG-OH was purchased from Sigma-Aldrich [BioUltra - grade; #95172 - poly(ethylene glycol) 20,000; LOT BCBF2828V]. Supplier provided molecular mass of as per certificate of analysis for the respective lots were used as such without any further cross verification. As per lot specific certificate of analysis, these macroinitiators were reported to have molecular mass of 19611 Da (based on hydroxyl value). All monomers and reagents were dried extensively by freeze drying process under high vacuum prior to transfer into the glove box.

Methods Polymer characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular weight determination by size exclusion chromatography (SEC) were conducted as described elsewhere.42 Hydrodynamic diameter (Dh) and zeta potential were determined as described elsewhere52 and each sample was measured 3 × and values are expressed average of these measurements. The desired pH of the micelle solution (for Figure 2 and 4) was obtained using 10 mM Britton-Robinson buffer. In vitro Dox release studies were conducted as described


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elsewhere.52 For the Dox release studies, 1 × PBS buffer (pH 7.4) or 150 mM sodium acetate buffer (pH 5) were used. Data for each release time point are average from 3 × independent experiments. In vitro cytotoxicity studies were conducted as described elsewhere52 and eight replicates were tested for one concentration of each sample. Stability studies of micelles in the presence of serum were evaluated as described elsewhere.54 Statistical analysis were performed as described elsewhere.55

Preparation of Doxorubicin-loaded micelles and measurement of loading Dox encapsulation was performed through a combination of sonication and (1) membrane filtration or (2) membrane dialysis method. For method 1, Dox (5 mg) (neutralized with 3 molar ratio of triethylamine) and polymer (10 mg) were dissolved with dimethyl sulfoxide (DMSO) (1 mL, 0.25 mL and 0.125 mL) respectively before mixing. The mixture was added dropwise into 10 mL HPLC-grade water while being sonicated at 130 W for 2 min by a probe based sonicator (Vibra Cell VCX 130). The solution was purified through filtration using a 2000 DA MWCO ultrafiltration spin column (Sartorius Stedim Vivaspin 15R) at 4000 rpm. Once the total volume of the solution has reached 1.5 mL, HPLC-grade water was used to top up the volume to 5 mL for 3 times. The resulting supernatant was removed and lyophilized. For method 2, after the sonication, the solution was dialysis against 1 L of DI water utilizing a dialysis bag with MWCO 1000 Da (Spectra/Por 7, Spectrum Laboratories Inc.). The water was changed at 3, 6, 24 h and resulting solution was collected and lyophilized at 48 h. To determine the amount of Dox in the lyophilized micelles, a known amount of the lyophilized sample was dissolved in 1 mL of DMSO and the absorbance of the solution was measured using a UV-Vis spectrophotometer (UV 2501PC, Shimadzu, Japan) at 480 nm. A calibration curve was obtained in the range of 1-100


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mg/mL with the r2 value of at least 0.99 to determine the Dox concentration. The reported Dox loading level are average of 3 × independent measurements and the Dox loading level was determined using the following formula: Actual loading level wt% =

100% −

Preparation of Diclofenac-loaded micelles and drug loading determination Diclofenac encapsulation was performed in various polymeric micelles through a combination of sonication and membrane filtration method. Diclofenac (2 mg) and polymer (10 mg) were dissolved with DMSO (0.125 mL) respectively before mixing. The mixture was added dropwise into HPLC-grade water (5 mL) under sonication for 10 mins using a 130 W probe based sonicator (Vibra Cell VCX 130). Unencapsulated diclofenac was removed via filtration using a 2000 Da MWCO ultrafiltration spin column (Sartorius Stedim Vivaspin 15R) at 4000 rpm. The mixture was concentrated to 1.5 mL and topped up to 5 mL for 3 times. The resulting supernatant was removed and lyophilized.

Drug loading determination The diclofenac loading content of diclofenac-loaded micelles was determined by high performance liquid chromatography (HPLC). Briefly, diclofenac-loaded micelles were dissolved in mixed solvent of acetonitrile and 0.0025 M sodium acetate in HPLC-grade water (70:30 by volume) to achieve a concentration of 1 mg/mL. The resulting solution (0.3 mL) was added into DMSO (0.7 mL) followed by sonication .The HPLC system consisted of a Waters 2690 separation module fitted with a Waters 996 Photodiode Array Detector and Waters XBridge C8 46 x 150 mm column. The elution rate was set at 1 mL/min and diclofenac detection wavelength


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was set at 276 nm. The column and sample temperature was maintained at 28 °C and 20 °C respectively. A calibration line was constructed to determine diclofenac concentration in the range of 1 to 100 mg/L, and the r2 value of peak area intensity plotted linearly against diclofenac concentration was at least 0.999. The reported DS loading level are average of 3 × independent measurements and the diclofenac loading level was calculated based on the following formula:

Actual loading level wt. % =

mass of diclofenac extracted from polymer x 100% mass of drug loaded polymer initially used

Polymer synthesis: Representative example (entry 2; Table 1): In a 7-mL vial containing a magnetic stir bar, 2a (211 mg, 954 µmol, 9.4 equiv.) and mPEG-OH (macroinitiator, 5.0 kDa, 506 mg, 101 µmol, 1.0 equiv.) were dissolved in DCM (~ 2.0 mL). To this solution, DBU (7.5 µL, 7.6 mg, 50.2 µmol., 1.0 equiv.) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature. After about 2 hours, the reaction was quenched by the addition of about 30 50 mg of benzoic acid. The reaction mixture was precipitated 3 times into diethylether: hexanes (80:20) mixture (50 mL) and the sample was dried under vacuum until constant mass was achieved, to obtain a white powdery solid.

Additional experimental notes on block copolymer synthesis: All polymerization reactions were conducted in nitrogen-filled glove box. Typically 5 mol% DBU, with respect to monomer, was used as the catalyst for N-aryl and N-carbamate type monomers (entries 1 - 3, 6 – 10, Table 1; polymerization time: 2 h). For N-alkyl type monomers either 40 mol% DBU (entries 4 and 5, Table 1; polymerization time: 24 h) or 5 mol % TBD


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(entry 11, Table 1; polymerization time: 2 h) with respect to monomer, was used as the catalyst. TBD was added from a DCM stock solution.

Synthesis of “all-carbonate” diblock copolymers via one-pot route: Synthesis of Bn-P(1a)-b-P(1c)-OH (6, entry 12, Table 1): In a 7-mL vial containing a magnetic stir bar, monomer 1a (112 mg, 540 µmol, 11.2 equiv.) and benzyl alcohol (5.0 µL, 5.2 mg, 48.3 µmol, 1.0 equiv.) were dissolved in DCM (1.0 mL). To this solution, DBU (3.6 µL, 3.7 mg, 24.2 µmol., 0.5 equiv.) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature for 2h. After 2h, an aliquot (60 µL) was removed to determine the monomer conversion (found to be ~ 99 % by 1H NMR spectroscopy). To the remaining reaction mixture a solution of monomer 1c (344 mg, 1330 µmol, 27.5 equiv. dissolved in 2.0 mL DCM) and additional DBU (54.1 µL, 55.1 mg, 362 µmol., 7.5 equiv.) were added and the reaction was allowed to proceed for another 44 h. The reaction was quenched by the addition of about 100 mg of benzoic acid. The crude polymer was purified by precipitating twice into cold diethylether (50 mL).

Synthesis of Bn-P(1a)-b-P(1d)-OH (7, entry 13, Table 1): In a 7-mL vial containing a magnetic stir bar, monomer 1a (103 mg, 497 µmol, 10.3 equiv.) and benzyl alcohol (5.0 µL, 5.2 mg, 48.3 µmol, 1.0 equiv.) were dissolved in DCM (1.0 mL). To this solution, DBU (3.6 µL, 3.7 mg, 24.2 µmol., 0.5 equiv.) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature for 125 min. After 125 min, an aliquot (60 µL) was removed to determine the monomer conversion (found to be ~ 99 % by 1H NMR spectroscopy). To the remaining reaction mixture a solution of monomer 1d (363 mg, 1570 µmol,


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32.5 equiv. dissolved in 2.0 mL DCM) and additional DBU (8.6 µL, 8.8 mg, 57.6 µmol., 1.2 equiv.) were added and the reaction was allowed to proceed for another 2 h. The reaction was quenched by the addition of about 100 mg of benzoic acid. The crude polymer was purified by precipitating twice into cold diethylether (50 mL).

Notes on deprotection of acid-sensitive protection groups: Deprotection of tert-butyl ester groups were conducted by subjecting the polymer to acidolysis (20 equiv. trifluoroacetic acid as 33 % solution in DCM) for ~ 1 h at room temperature, in a loosely capped scintillation vial (20 mL). The polymer is first dissolved in DCM and to this solution, TFA was added. The crude polymer was purified by precipitating into cold diethylether (50 mL). Likewise, deprotection of tert-butyloxycarbonyl groups, were conducted by subjecting the polymer to acidolysis (20 – 30 equiv. trifluoroacetic acid either neat or as ~ 85 % solution in DCM) for ~ 1 h at room temperature in a loosely capped scintillation vial (20 mL). The crude polymer was purified by precipitating into cold diethylether (50 mL).


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Results and Discussion The commercial availability of several diethanolamine derivatives and chemo-selective functionalization of diethanolamine, paves way for the step-efficient synthesis of N-substituted 8-membered aliphatic cyclic carbonates.42 With a hydrophilic macro-initiator such as poly(ethylene glycol) (PEG) of initiating hydroxyl functionalities either at the ω- or at both αand ω- chain ends, amphiphilic block copolymers can be readily obtained. The introduction of PEG brings in numerous advantages including aqueous stability due to the hydrophilicity and “stealth”- character56 to the resultant self-assembled nanostructures so that the cargos are not recognized and taken up by the reticuloendothelial systems.57 In this study, a series of poly(ethylene oxide-b-carbonate) copolymers were designed and synthesized having different carbonate-block compositions, architectures and sequences (Table 1, Scheme 1).

The poly(ethylene oxide-b-carbonate) copolymers were prepared by initiating the polymerization of 1a using poly(ethylene glycol) monomethyl ether (mPEG-OH, 5.0 kDa) as the macroinitiator, in the presence of 5.0 mol % DBU (relative to the monomer) in DCM, to generate the requisite AB diblock copolymers, 2a1 - 2a3. Narrowly dispersed products (ÐM) were obtained with predictable molecular weights by changing the initial feed ratio (monomer to initiator ratio) (Table 1, entries 1-3). Using these general conditions, it was possible to polymerize different monomer combinations to generate AB or A(BC) type diblock copolymers (Table 1, entries 6 – 9). The copolymerization of functional cyclic carbonates offers a simple handle to tailor the coreenvironment of the resultant self-assembled nanostructures and has been shown to have a tremendous influence in kinetic stability and release rate profiles in drug carriers.4 Additional reactive sites were introduced with allyl groups (entry 9) that can be used for further


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functionalization through post-polymerization reactions10, 21 or for post-assembly cross-linking.58 Poly(carbonate-b-ethylene oxide-b-carbonate) (BAB) triblock copolymers were synthesized with monomer 1a by using a dihydroxyl functional PEG (Scheme 1, Table 1, entry 10) as the initiator. This BAB architecture is capable of forming “flower-shaped” or “star-shaped” micelles in aqueous solution, which can form hydrogels at higher concentrations.59-62


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Scheme 1. Synthetic routes to access PEGylated amphiphilic block copolymers with different configurations: (A) Well-defined diblock copolymers of different compositions such as AB and A(BC) can be accessed through the choice of the functional monomer and through copolymerization of two different monomers respectively, to tailor the physico-chemical properties of hydrophobic “core-forming” block for specific applications. Materials with tunable inter-particle associative interactions, capable of forming hydrogels, can be accessed with BAB triblock copolymers; (B) Structures of functional 8-membered N-substituted aliphatic cyclic carbonate monomers and the organo-catalysts used for synthesis of the AB, A(BC) and BAB block copolymers.


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Previously we found that the catalyst, concentration and nature of N-substitution dictate the polymerization kinetics of these 8-membered aliphatic cyclic carbonates.42 For example, the Naryl and N-carbamate monomers polymerized significantly faster than the N-alkyl class of cyclic carbonates. However, increasing the amount of DBU (40 mol % relative to the monomer) or changing catalyst from DBU to TBD significantly increased the polymerization rate of N-alkyl substituted monomers.42 Hence, this approach was used to polymerize the N-alkyl class of cyclic carbonates, 1b and 1c to access AB and BAB type block copolymers (Table 1, entries 4, 5 and 11).

“All degradable carbonate” diblock (BC) copolymers (poly(carbonate-b-carbonate)) were also prepared in a one-pot sequential polymerization. Since monomer 1a would lead to a relatively hydrophobic block and also high monomer conversion can be achieved, it was chosen as the first block. Polymerization of monomer 1a was initiated with benzyl alcohol and when the growth of first block approached completion (monomer conversion ~ 99%), the second monomer, 1c or 1d, was added to generate BC diblock copolymers 6 and 7, respectively (Scheme 2, Table 1, entries 12 and 13). A combination of size exclusion chromatography and 1H NMR spectroscopy were used to characterize all these polymers (Table 1, Figure S1 – S28). The results demonstrate that well-defined block copolymers with relatively low molar dispersity (ÐM) were obtained from these N-substituted 8-membered aliphatic cyclic carbonates.


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Scheme 2. Synthetic routes to access “all-carbonate” diblock copolymers (BC) that served as amphiphilic block copolymer precursors. Upon deprotection of tert-butyl esters (6) or tBocprotection groups (7), amphiphilic block copolymers were produced.

Post-polymerization deprotection of tert-butyl ester and tert-butyloxycarbonyl residues in the presence of trifluoracetic acid resulted in the cationic (Scheme 3B) or zwitterionic (Scheme 3C) block copolymers. Polymers transformed by acidolysis are represented with their original code, followed by an apostrophe. For example, the double hydrophilic polymer from 2d is represented as 2d’. Absence of resonances associated with the tert-butyl protection groups in 1H NMR spectroscopy, indicated successful deprotection (Figure S29 – S32), resulting in hydrophilic cationic polymer (2d’) and amphiphilic block copolymers (3d-e’, 6’ and 7’). These block copolymers form micelles and hydrogels, and have various potential applications. Following are a few examples.


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Micelles formed from copolymers containing pH-responsive tertiary amines and their use for hydrophobic drug delivery Polycarbonates containing tertiary amines were synthesized using N-alkyl substituted 8membered aliphatic cyclic carbonates. Under acidic conditions the tertiary amine in the backbone can pick up protons leading to the formation of quaternary ammonium salts (Scheme 3A). This pH-responsive transformation is useful in drug delivery as it can facilitate drug release in acidic endolysosomes following cellular uptake, improving therapeutic efficacy. This concept was utilized to deliver a model anticancer drug, doxorubicin. Polymer 2b was specifically chosen for the following reasons: (1) the benzyl group and tert-amine group in the polymer can offer hydrophobic environment that is capable of participating in π−π and hydrogen-bonding interactions with the drug, respectively; (2) the tert-amine in the hydrophobic polycarbonate block can undergo protonation in an acidic environment, enabling for the change in the amphiphilic balance of the block copolymers, and thus allowing for the enhanced release of Dox at a relatively lower pH. Since it has been well-documented that both the tumor microenvironment and the intra-cellular organelles such as endosomes/lysosomes are acidic, increased Dox release in these environments could improve anticancer efficacy and reduce non-specific toxicity in healthy tissues; (3) the diblock copolymer design with PEG as the hydrophilic block enables aqueous stability and at the same time incorporates “stealth” for prolonged blood circulation.50, 56-57


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Scheme 3. Schematic representation of pH-responsive polycarbonates. (A) Protonation of tertiary amine to a cationic quaternary ammonium salt; (B) acidolysis allowed for access to polycarbonate containing secondary amine functionality and (C) pH-responsive zwitterions that could be modulated in principle to access not only zwitterionic, but also, cationic, anionic and charge-neutral configurations under different pH environments.


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Table 1. Synthesis and characterization of aliphatic N-substituted 8-membered cyclic carbonates-derived block copolymers

Codea

Type

Description

DPn b, d (A)

DPnc, d (B)

DPnc, d (C)

Mn (kDa)d

1

2a-1

AB

mPEG-b-P(1a)-OH

113

5

-

6.0

1.09

2

2a-2

AB

mPEG-b-P(1a)-OH

113

10

-

7.1

1.11

3

2a-3

AB

mPEG-b-P(1a)-OH

113

19

-

8.9

1.14

4

2b

AB

mPEG-b-P(1b)-OH

113

17

-

8.8

1.13

5

2c

AB

mPEG-b-P(1c)-OH

113

12

-

8.1

1.11

6

2d

AB

mPEG-b-P(1d)-OH

113

19

-

9.4

1.12

7

2e

AB

mPEG-b-P(1e)-OH

113

22

-

9.5

1.09

8

3d-e

A(BC)

mPEG-b-P(1d-co-1e)-OH

113

10

10

9.3

1.11

9

3d-f

A(BC)

mPEG-b-P(1d-co-1f)-OH

113

40

5

15.3

1.24

10

4a

BAB

HO-P(1a)-b-PEG-b-P(1a)-OH

445

9

-

23.3

1.20

11

4b

BAB

HO-P(1b)-b-PEG-b-P(1b)-OH

445

10

-

23.6

1.15

12

6

BC

Bn-P(1a)-b-P(1c)-OH

-

12

16

6.6

1.17

S. No.

(NMR)

ÐM e

13 Bn-P(1a)-b-P(1d)-OH 10 26 8.1 1.14 7 BC a – Please refer to the schemes for the code description; b – denoting degree of polymerization (DPn) of the initiating macroinitiator block (as provided by the supplier) or the first block grown off from the small molecule initiator (as determined by 1H NMR spectroscopy); c – denoting DPn of the first block grown off from the macroinitiator or the second block grown from the aliphatic functional polycarbonate-based macroinitiator – both as determined by 1H NMR spectroscopy; d - determined by 1H NMR spectroscopy; e – uncorrected; determined by size exclusion chromatography (SEC) in THF, column calibrated by using polystyrene standards.


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Dox was loaded into polymer 2b micelles via self-assembly. Preparation conditions were optimized to produce micelles having nanosize and high drug loading level. Specifically, Dox (5 mg) and polymer (2a, 10 mg) were dissolved each in DMSO (2.0 mL) and added dropwise into water (10 mL) under sonication for 10 min. DMSO and unencapsulated Dox were subsequently removed either using a dialysis or centrifugal filtration method. The Dox-loaded 2b, obtained via the centrifugal filtration method was found to have higher Dox loading contents and also relatively smaller hydrodynamic diameters. Encouraged by these findings, the encapsulation protocol was further refined with the reduction of DMSO from 2.0 mL to 0.25 mL that dramatically increased Dox loading content from 8.7 ± 0.8 wt.% to 33.0 ± 2.7 wt.% (Figure 1B). The hydrodynamic diameter of these drug-loaded nanoparticles as measured by dynamic light scattering (DLS) technique were found to be 113 ± 8 nm (intensity average) with a relatively narrow PDI (0.23 ± 0.03). Although polymeric micellar carriers with high Dox loading contents (> 30 wt.%) were reported before, many of these systems were either non-degradable or involved multi-step polymer synthesis to access the requisite functional groups.27, 52 In contrast, polymer 2b can be obtained from relatively inexpensive starting materials in a highly step- and atom-efficient manner. In vitro Dox release was found to be pH-sensitive, and Dox release was faster at the endolysosomal pH (pH 5.0) than pH 7.4 of healthy tissues and blood stream (Dox release: 65 % vs. 50 %) (Figure 1C). Furthermore, Dox-loaded 2b micelles exerted a cytotoxic effect against HepG2 human liver cancer cell line, comparable to free Dox, with an IC50 value (Dox concentration that causes 50% inhibition in cancer cell growth) of ~ 0.3 µg/mL (Figure 1D), while polymer 2b showed negligible toxicity towards both HepG2 and HEK 293 human embryonic kidney cell lines (Figure 1E). The


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results demonstrated potential of these tertiary amine-containing polymers as versatile drug delivery carriers.


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Figure 1. Evaluation of polymer 2b as a drug delivery vehicle for the model anticancer drug doxorubicin (Dox): (A) Chemical structure of polymer 2b and Dox; (B) Encapsulation method dependent hydrodynamic diameter of the Dox-encapsulated polymer 2b nanoparticles and Dox loading contents. P < 0.01, 2 mL DMSO centrifugal filtration against 2 mL DMSO dialysis. P < 0.01, 0.5 mL DMSO against 2 mL DMSO. P < 0.05, 0.25 mL DMSO against 0.5 mL DMSO; (C) In vitro cumulative Dox release profiles from 2b nanoparticles at pH = 7.4 (squares) and pH = 5.0 (circles). P < 0.05, for all time point ≥ 2 h for pH 5.0 against 7.4.; (D) Viability of HepG2 cells after incubation with free Dox (squares) and Dox-loaded nanoparticles (circles) at various Dox concentrations and (E) Viability of HEK 293 (squares) and HepG2 (circles) cells after 48 h of incubation with polymer 2b at various concentrations.

Micelles formed from copolymers containing pH-responsive secondary amines for hydrophilic drug delivery t

Boc-protected secondary amine-containing polycarbonates were prepared from N-tBoc

substituted 8-membered aliphatic cyclic carbonate monomer (1d). Post-polymerization deprotection of the tBoc-protection groups under acidolysis conditions resulted in polymers containing secondary amine across the backbone. Typically the deprotection was carried out under excess of acid, leading to a protonated quaternary ammonium salt that could reversibly respond to pH changes (Scheme 3B). To demonstrate the pH-responsiveness, the diblock copolymer 7’ (Figure 2A), which was prepared by the deprotection of polymer 7, was used. Under acidic conditions (pH < 7.0), the self-assembled nanostructures had a cationic shell and at pH > 7.0, the shell became almost neutral (Figure 2B). The pH-sensitivity was also evidenced by change in particle size. The polymer formed stable nanostructures of ~ 100 nm in


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size under acidic conditions, while aggregation was found at pH > 7.0 stemming from neutral surface (Figure 2B).

Figure 2. pH-responsive secondary amine-containing polycarbonate. (A) amphiphilic block copolymer 7’ with a cationic hydrophilic block containing secondary amines; (B) pHdependent zeta-potential and hydrodynamic diameter of self-assembled nanostructures from 7’.

Three different PEG/secondary amine-functionalized polycarbonate diblock copolymers – 2d’, 3d-e’ and 2e were synthesized from the same mPEG-OH macro-initiator (5.0 kDa), but the composition of the polycarbonate block was varied to elucidate the role of hydrophobicity and


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cationic charge on the encapsulation of an anionic model drug, diclofenac sodium salt (DS, a non-steroidal anti-inflammatory drug-NSAID) (Figure 3A). Encapsulation of DS into these polymeric carriers was conducted in a procedure similar to that of optimized Dox loading experiments by using 0.25 mL of DMSO (Figure 3B). DS and respective polymer were used in a ratio of 2 mg : 10 mg. With 2d’ containing the cationic-residues alone, the solutions precipitated. However for the polymers containing hydrophobic residues (3d-e’ and 2e), stable solutions were observed. The intensity average hydrodynamic diameter of these DS-loaded nanoparticles as measured by DLS was found to be larger for the DS-3d-e’ (163 ± 21 nm) than DS-2e (29 ± 1 nm) with a relatively narrow PDI (< 0.20). Likewise the DS loading content was dramatically higher for the DS-3d-e’ (12.5 ± 0.1 wt. %) in comparison to DS-2e (0.8 ± 0.1 wt. %). The final DS loading content increased to 20.5 ± 0.2 wt. % when the initial DS to polymer weight ratio was increased to 5:10. Loading contents for 3d-e’ based formulations are ~ 2 – 3 times higher than the values reported in literature for methoxypoly(ethylene glycol)poly(ε-caprolactone) based micelles (5.8 ± 0.8 wt. %),63 suggesting that both the cationic and hydrophobic environments are necessary for the successful incorporation of DS. These micelles may also be used to deliver other anionic drugs including antibiotics via ionic interaction.

pH-responsive zwitterionic polymers as PEG replacements Zwitterions are particularly exciting due to their antifouling behavior, and offer an attractive alternative to PEG. Antifouling materials have been used as implant coatings to improve biocompatibility, and as the shell of drug delivery nanocarriers for prolonged blood circulation.51,

64-65

Zwitterions-containing polycarbonates were obtained from monomer 1c


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upon post-polymerization deprotection of the tbutyl ester protection groups under acidolysis conditions (Scheme 3C). Flexibility to modulate these polymers to switch from cationic, to neutral or zwitterionic to anionic molecules (Scheme 3C), renders them attractive for a wide variety of applications. To investigate the pH-responsiveness, the diblock copolymer 6’ (Figure 4A) was prepared by the deprotection of polymer 6. The zeta-potential and the intensity average hydrodynamic diameter of the self-assembled nanostructures formed by this polymer were investigated under different pH conditions (Figure 4B). As a function of solution pH, the zeta-potential of the self-assembled nanostructures was found to have three distinct zones corresponding to cationic, neutral, zwitterionic or anionic shell. Under acidic conditions of pH < 5.0, the self-assembled nanostructures have a cationic shell. At intermediate pH values of 5.0 to 7.4, nanostructures were found to have a neutral shell and at pH values ≥ 7.5, anionic shell was observed. Unlike zeta-potential, the particle size was found to be stable at ~ 30 nm, independent of the environmental pH. To determine the non-fouling nature of these nanostructures, we investigated the hydrodynamic size of these nanostructures as a function of time in a serum-containing medium. These nanostructures were found to be relatively stable in PBS buffer (pH 7.4) containing 10% fetal bovine serum (Figure S33). These results demonstrate the potential utility of these hydrophilic zwitterionic polycarbonates for antifouling applications.64-65


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Figure 3. PEG and secondary amine-containing polycarbonate diblock copolymers for delivery of the model anionic drug diclofenac sodium salt (DS). (A) Chemical structure of polymers 2d’, 3d’-e and 2e, representing building blocks that would result in nanostructures with an entirely ionic, a combination of both ionic and hydrophobic and entirely hydrophobic core respectively, structure of DS along with the plausible interactions between the block copolymer and the drug; (B) Hydrodynamic diameter, PDI and DS loading contents of DS-encapsulated nanoparticles formed from various polymers at different polymer to DS feed ratios. P < 0.01, for 3d-e’ against 2e and for 3d-e’ (10:2) against 3d-e’ (10:5).


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Figure 4. pH-responsive zwitterionic polymer. (A) amphiphilic block copolymer 6’ with a zwitterionic hydrophilic block; (B) pH-dependent zeta-potential and hydrodynamic diameter of self-assembled nanostructures from 6’.

Hydrogels formed from amphiphilic triblock copolymers BAB-type triblock copolymers in a middle-block selective solvent can self-assemble in a concentration dependent manner to form a variety of self-assembled nanostructures.59 Typically as the concentration of the triblock copolymers is increased they have propensity to form gels via inter-micellar association. Triblock copolymers 4a and 4b (Table 1), with a


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hydrophilic PEG middle block and hydrophobic end-blocks were synthesized from carbonate monomers 1a and 1b, respectively. These poly(carbonate) end-blocks are relatively hydrophobic (in comparison to PEG) and contain pH-responsive tertiary amines along the backbone. As evidenced from vial inversion test, at 5.0 wt. %, both triblocks 4a and 4b formed hydrogels under neutral aqueous conditions but existed as solution under acidic conditions (Figure 5). Compared to some of the existing triblock copolymer based gelators, it is remarkable that 4a and 4b can form pH-responsive gel at a relatively low polymer concentration and hydrophobic weight fraction. For instance, in contrast to our system (5.0 wt. % triblocks and hydrophobic weight fraction of ~ 0.16 and 0.17 for 4a and 4b, respectively), ~ 20 wt.% of poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) triblock copolymer with hydrophobic weight fraction of ~ 0.30 was necessary to form hydrogel.66 The observed pH-dependent gel-to-solution transition is due to protonation of amines present across the backbone of polycarbonate end-blocks (and consequent charge-charge repulsion). These hydrogels could potentially be used as topical and injectable drug delivery formulations.67


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Figure 5. pH-responsive hydrogels formed by BAB triblock copolymers 4a and 4b.

Conclusion Well-defined biodegradable block copolymers consisting of pH-responsive residues such as tertiary amines, secondary amines and zwitterions were synthesized from diethanolaminederived functional eight-membered N-substituted aliphatic cyclic carbonates either directly via ROP (tertiary amines) or through the combination of ROP and post-polymerization deprotection strategy (secondary amines and zwitterions). Given availability of inexpensive starting materials, step-efficient routes to access functional monomers and excellent control over numerous aspects of polymerization, this approach allows for access to functional biodegradable polymers for various biomedical applications. Particularly, pH-responsive diblock copolymers containing secondary and tertiary amines have been demonstrated to be promising as delivery carriers for the hydrophobic (Dox) and hydrophilic (DS) drugs.


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Preliminary promising antifouling and non-toxic results suggest that zwitterionic polycarbonates may be used as a biodegradable alternative to PEG for therapeutic delivery. Therefore, this novel series of biodegradable functional polycarbonates is promising for nanomedicine.

Supporting Information NMR and SEC data for all the reported block copolymers and stability of micelles from polymer 6’ in the presence of serum

Acknowledgements This work was funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), and IBM Almaden Research Center, U.S.A.

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Amphiphilic and Hydrophilic Block Copolymers from Aliphatic N

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