Well-Defined Degradable Brush PolymerâDrug Conjugates for

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Well-Defined Degradable Brush Polymer-Drug Conjugates for Sustained Delivery of Paclitaxel Yun Yu, Chih-Kuang Chen, Wing-Cheung Law, Jorge Mok, Jiong Zou, Paras N Prasad, and Chong Cheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp3004868 • Publication Date (Web): 26 Nov 2012 Downloaded from http://pubs.acs.org on December 7, 2012

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Molecular Pharmaceutics

Well-Defined Degradable Brush Polymer-Drug Conjugates for Sustained Delivery of Paclitaxel Yun Yu,† Chih-Kuang Chen,† Wing-Cheung Law,‡ Jorge Mok,† Jiong Zou, †,§ Paras N. Prasad,‡ and Chong Cheng*, †

†

Department of Chemical and Biological Engineering, ‡Institute for Lasers, Photonics and

Biophotonics, the State University of New York at Buffalo, Buffalo, NY 14260.

KEYWORDS: polymer-drug conjugate, drug delivery, brush polymer, click chemistry, polylactide, paclitaxel

ACS Paragon Plus Environment

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ABSTRACT: To achieve a conjugated drug delivery system with high drug loading but minimal long-term side effects, a degradable brush polymer-drug conjugate (BPDC) was synthesized through azide-alkyne click reaction of acetylene-functionalized polylactide (PLA) with azidefunctionalized paclitaxel (PTXL) and poly(ethylene glycol) (PEG). Well-controlled structure of the resulting BPDC and its precursors were verified by 1H NMR and gel permeation chromatography (GPC) characterizations. With nearly quantitative click efficiency, drug loading amount of the BPDC reached 23.2 wt%. Both dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM) imaging indicated that the BPDC had a nanoscopic size around 10-30 nm. The significant hydrolytic degradability of the PLA backbone of the BPDC was confirmed by GPC analysis of its incubated solution. Drug release study showed that PTXL moieties can be released through the cleavage of the hydrolysable conjugation linkage in pH 7.4 at 37 ºC, with 50% release in about 22 h. As illustrated by cytotoxicity study, while the polymeric scaffold of the BPDC is non-toxic, the BPDC exhibited higher therapeutic efficacy towards MCF-7 cancer cells than free PTXL at 0.1 and 1 µg/mL. Using nile red as encapsulated fluorescence probe, cell uptake study showed effective internalization of the BPDC into the cells.


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Introduction

Many biologically active drugs are limited in clinical use due to their poor solubility, nonspecific uptake and rapid elimination.1 For instance, as a potent anti-cancer drug, paclitaxel (PTXL) can remarkably improve patient survival time,2 but the very poor water solubility of PTXL (0.00025 mg/mL) is a critical problem for intravenous administration. Cremophor EL and other organic solvents (such as ethanol) have been used to solubilize PTXL for delivery, but unfortunately they can induce significant side effects.2-4 Moreover, the circulation times of small molecular drugs, including PTXL and many others, are very short in blood stream, and the bioavailability of these drugs typically is low.1 Polymeric nanoparticulate drug delivery systems have been widely developed over the past decade.5-7 Relative to small molecule drugs, they may have prolonged circulation time, reduced non-specific uptake and side effects, improved water solubility and increased accumulation in tumor tissue through enhanced permeation and retention (EPR) effect. As an important type of drug delivery system, polymer-drug conjugate (PDC) possesses drug moieties covalently bonded to polymer scaffolds through linkages that may be cleaved for drug release under desirable circumstances.1,8 As compared with drug encapsulation systems using assembled or aggregated scaffolds, PDCs typically have better stability against environmental changes. Moreover, PDCs with well-defined structures generally have precise drug loading amounts and can provide sustained drug release without burst effect.8 The structural design of base polymers of PDCs requires thoughtful considerations. Linear polymers have been broadly studied as the base polymers of PDCs.1 However, drug moieties may not be readily shielded in the resulting PDCs, and significant peripheral hydrophobicity of


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these PDCs would induce unfavorable aggregations. Therefore, optimal drug loadings in these PDCs typically are relatively low.1

Moreover, polymers that are non-degradable under

biological aqueous conditions may cause long-term toxicity. To dissipate the concern of longterm side effects, a general strategy is to limit MWs (≤ 45 kDa) of the base polymers.6 However, even complete renal clearance may be realized for the base polymers with relatively low MWs, the circulation time of the corresponding PDCs can be significantly restricted. As an important class of biodegradable and biocompatible polymers, aliphatic polyesters have been broadly studied for therapeutic delivery applications.9-15 In most cases, aliphatic polyesters were used for drug encapsulation via nanoparticles or polymeric micelles.16-20

Studies on

aliphatic polyester-based PDCs were rather limited, mainly due to the lack of functionality and hydrophilicity of typical polyesters. Recently, some important progresses have been made in the preparation and studies of aliphatic polyester-based PDCs.21-26

Cheng and co-workers

synthesized PDCs with drug moieties at α-terminals of polylactides (PLAs) by ring-opening polymerization (ROP) of lactide using drugs as initiators, and further obtained water-dispersible nanoparticles from these PDCs by nanoprecipitation.21,22 PDCs using multivalent base polymers may possess improved drug loading amounts. Emrick and co-workers prepared a water-soluble PDC with multiple pendent camptothecin moieties and poly(ethylene glycol) (PEG) side-chains through click chemistry via an alkyne-functionalized polyester.23

Fréchet and co-workers

synthesized bow-tie PDCs with a polyester dendrimer core conjugated with multiple doxorubicin (Dox) moieties and PEG chains.24

With well-defined nanostructures and hydrophilic PEG

component, these dendrimer-Dox conjugates further exhibited long circulation time and high tumor uptake in mice bearing C-26 colon carcinomas.


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With interesting nanostructures, brush polymers have been utilized for applications in therapeutic delivery.27,28 Representing a new class of PDCs, brush polymer-drug conjugates (BPDCs) have been reported by both Johnson et al. and us.29-31 In the structural design of these BPDCs, hydrophobic drug moieties were covalently linked with the backbone of peglyated brush polymers through cleavable linkages. With well-shielded environments for drug moieties and minimal peripheral hydrophobicity, these BPDCs can possess not only relatively high drug loading efficiency but also suppressed unfavorable aggregation and non-specific membrane binding. Their PEG-based grafts may further help to promote water-solubility and circulation time of these BPDCs.32 However, the first examples of BPDCs were prepared through ringopening metathesis polymerization, and therefore, with non-degradable polynorbornene-based backbones and high MWs, the polymer scaffolds may not be readily eliminated from biological systems after drug release. In order to achieve BPDCs with minimal long-term side effects for potential clinical applications, we have studied biodegradable BPDCs with PLA backbones.25 As we reported recently, a well-defined biodegradable BPDC with PTXL drug moieties as bivalent agents to bridge between PLA backbone and PEG side chains was successfully obtained by the coppercatalyzed azide–alkyne cycloaddition reaction of acetylene-functionalized PLA with azidefunctionalized PTXL–PEG conjugate. However, drug release from this BPDC cannot be readily controlled, because it requires the successive cleavages of two types of conjugation bonds with different sensitivities. To facilitate drug release, it is preferred that drug moieties serve as monovalent agents to link with delivery scaffolds. Therefore, very recently we synthesized and studied a BPDC with PLA backbone linked respectively with PTXL moieties and PEG chains.


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In this article, we report the synthesis, characterization, and in vitro studies of such a biodegradable BPDC.

Results and Discussion

Synthesis and Characterization of BPDC. Because of the high efficiency of azide-alkyne click reaction,33 grafting-onto synthesis of PLA-g-PTXL/PEG BPDC via azide-alkyne chemistry was designed (Scheme 1).

Accordingly, alkyne and azide-functionalized precursors of the

BPDC need to be prepared at first. Using acetylene-functionalized lactide (1) as monomer, ethanol

as

the

initiator

and

4-dimethylaminopyridine

(DMAP)

as

catalyst

([1]0:[EtOH]0:[DMAP]0 = 60:1:4), acetylene-functionalized PLA (2) was obtained by ringopening polymerization (ROP) in dichloromethane (DCM) at 35 ºC for 24 h.25 As revealed by 1

H NMR analysis of the final polymerization solution, the monomer conversion reached 90%.

After precipitation of the polymerization solution in ice-cold methanol, the resulting 2 was analyzed by 1H NMR spectroscopy (Figure 1a) and GPC (Figure 2a), and its well-controlled chemical structure was verified. The number-average degree of polymerization (DPn) of 2 was determined as 54 by comparing the resonance intensities of the CH protons from 1 at 5.26-5.35 ppm with these of the terminal CH2 protons from ethanol at 4.21 ppm.25 This experimental DPn agreed precisely with the DPn calculated from feed ratio and monomer conversion, indicating the living characteristic of the ROP process.34 According to the DPnNMR value, the number-average molecular weight (Mn) of 2 was further determined as 9.1 kDa. As characterized by GPC analysis, 2 had a Mn of 14.4 kDa and a PDI of 1.15 relative to linear polystyrenes. The low PDI value of 2 also indicated the excellent synthetic control in the polymerization.


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Scheme 1. Synthesis of PLA-g-PTXL/PEG BPDC 6

Figure 1. 1H NMR spectra of acetylene-functionalized PLA 2 (a), azide-functionalized PTXL 4 (b), and PLA-g-PTXL/PEG BPDC 6 (c)


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Figure 2. GPC curves of acetylene-functionalized PLA 2 (a), the aliquots of click reaction after 1 h (b), 3 h (c), 5 h (d) and 20 h (e), and PLA-g-PTXL/PEG BPDC 6 (f).

Azide-functionalized PTXL and PEG were also prepared respectively. Catalyzed by N,N'dicyclohexylcarbodiimide (DCC) and DMAP, esterification reaction of 2-(2-(5-azidopentyl)-5oxo-1,3-dioxol-4-anyl)acetic acid (3) with PTXL was conducted in DCM at room temperature for 24 h ([3]0:[PTXL]0:[DCC]0:[DMAP]0 = 1:1:1.5:0.2).25 Although both hydroxyl groups at 2’ and 7 positions of PTXL are chemically reactive, the 2’-hydroxyl group typically exhibits much higher reactivity than the 7-hydroxyl group.2 Therefore, azide-functionalized PTXL (4) with functionality at 2’ position was obtained in 76% yield. The chemical structure of 4 was verified by 1H NMR analysis (Figure 1b). In the 1H NMR spectrum of 4, the characteristic resonance of 7-CH at 4.45 ppm remained, while the resonance of 2’-CH proton were shifted from 4.78 ppm


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(for reactant PTXL) to 5.46 ppm (for 4). The characteristic resonances of the protons a-c from the cycloacetal group in 4 were also identified. The resonance intensities of these protons were compared with that of 5-CH proton of PTXL at 4.98 ppm, and the resulting ratio of the resonance intensities agreed very well with the corresponding proton ratio in 4. α-Methoxyl ωazido PEG2000 (5; DP = 45) was prepared from α-methyloxy PEG2000 via functional group transformation, following a literature approach.35 The BPDC 6 was successfully synthesized by the azide-alkyne click reaction of 4 and 5 with 2 in DMF at r.t., in the presence of copper sulfate pentahydrate and sodium ascorbate ([acetylene of 2]0:[4]0:[5]0:[CuSO4•5H2O]0:[NaAsc]0 = 1.0:0.5:0.5:0.05:0.12).36 The reaction process was monitored by GPC analysis of the reaction aliquots (Figure 2). The peak retention volume of 2 was 17.4 mL, as shown in the curve a. After the click reaction was conducted for 1 h (curve b), the GPC peak of 2 essentially disappeared but a new peak of grafted product was observed at 15.7 mL, while the peaks of 5 at 19.2 mL and 4 at 20.6 mL were still significant, indicating the limited extent of graft-onto reaction at that time. With the increase of reaction times (curve c: 3 h; curve d: 5 h), the peak heights of 4 and 5 reduced and the new peak of grafted product shifted towards higher molecular weight side. After 20 h (curve e), the peaks of 4 and 5 became very small and new peak position at 15.1 mL kept unchanged, indicating that the click efficiency for the grafting-onto process reached ~95% and the hydrodynamic volume of the resulting densely grafted product was insensitive to further increase of grafting density.

The reaction was

eventually stopped at 24 h, and the resulting BPDC 6 was obtained in 60% yield after passing the final reaction solution through a short alumina column to remove catalysts (using CH2Cl2 as eluent), followed by precipitation in diethyl ether.

1

H NMR analysis verified the chemical

structure of the BPDC. Relative to the LA-based backbone units, 46.5% grafting of PTXL


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moieties and 51.6% grafting of PEG chains were determined for 6 by comparing the resonance intensities of the 5-CH proton from PTXL at 4.97 ppm and the CH2 protons of PEG at 3.49-3.83 ppm with the resonance intensity of the CH protons of functional LA units at 5.13 ppm. Thus, 6 had a MnNMR of 92.2 kDa, with 23.2 wt% of PTXL. This PTXL loading amount in 6 was much higher than the PTXL loading in Abraxane (~10 wt%), a FDA-approved protein-bound PTXL clinical therapy for breast cancer treatment.37 BPDC 6 showed a mono-modal GPC peak (curve f, Figure 2), corresponding to a Mn of 68.7 kDa and a PDI of 1.16 relative to linear polystyrenes. Its MnGPC was smaller than its MnNMR, as a result of its densely grafted compact structure.38

Figure 3. DLS size distribution profiles of BPDC 6 in water and in DMF (a); a TEM image of BPDC 6 (b).

Due to the hydrophilicity of the grafted PEG chains of 6, the water solubility of 6 is excellent. It can be quickly dissolved in water without any external assistance to give aqueous solutions. The size and size distribution of 6 in water and in DMF were measured by dynamic light scattering (DLS) analysis (Figure 3a). It had a hydrodynamic size around 10-30 nm, with a volume-average hydrodynamic diameter (Dh,v) of 15.5 ± 5.0 nm at 1.0 mg/mL in water. Because 6 had a larger hydrodynamic size in water than in DMF (Dh,v = 10 ± 2 nm), which is a good


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solvent for all of its components, the occurrence of assembling of 6 in water was suggested. Having a good agreement with the DLS results, TEM imaging showed that elliptical nanoparticles of 6 with size around 10-30 nm were obtained by dip coating an aqueous solution of 6 on a TEM grid (Figure 3b).

Because kidney and reticuloendothelial clearance can

effectively clear nanostructures with smaller sizes (

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