CROSS-LINKED POLYMER MICELLES WITH BIODEGRADABLE IONIC CORES FOR ANTI-CANCER DRUG DELIVERY

Introduction

Structural decay of the micelles under severe dilution in the blood compartment, leading to premature drug release, limits the use of polymer micelles as carriers for improved drug delivery. Intramicellar cross-linking is one of the promising methods to improve the stability of polymeric micelles against dilution, ionic strength, temperature and shear forces ^1–4. In particular, stabilization of multimolecular micelle structure using disulphide containing cross-linkers is particularly attractive because of the differences in the reductive potential outside and inside of the cell. While disulphide containing carrier systems possess high stability in the systemic circulation and in extracellular fluids, they are prone to rapid degradation under a reductive environment present in the intracellular compartment, allowing controlled cytoplasmic delivery of the bioactive payload⁵.

In this study, block ionomer complexes (BIC) of poly(ethylene oxide)-b-poly(methacylic acid) (PEO-b-PMA) and divalent metal cations (Ca²⁺) were utilized as templates for the synthesis of cross-linked (cl)-micelles^1–2. Cystamine was utilized as a biodegradable cross-linker. Doxorubicin (DOX) or cisplatin (CDDP) was loaded into the cross-linked core of the polymeric micelles. The physicochemical properties, loading efficacy, the release of DOX or CDDP from the polymer micelles with biodegradable cross-links in the core as well as in vitro cytotoxicity were investigated.

Experimental

Materials

Poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMA) diblock copolymer (M_w/M_n = 1.45) was purchased from Polymer Source Inc., Canada. The block lengths were 170 and 180 repeating units for PEO and PMA, respectively. Cisplatin, calcium chloride, cystamine dihydrochloride, ethylenediamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich (St Louis, MO). Doxorubicin hydrochloride was a kind gift from Dong-A Pharmaceutical Company, South Korea. All other chemicals were of reagent grade and used without further purification.

Synthesis of Cross-linked Polymer Micelles

cl-micelles was prepared by using BIC of PEO-b-PMA copolymer and divalent metal cations as templates by the previously described method with a slight modification^1–2. In brief, PEO-b-PMA/Ca²⁺ complexes were prepared by mixing an aqueous solution of PEO-b-PMA with a solution of CaCl₂ at a molar ratio of [Ca²⁺]/[COO⁻]=1.3. EDC was added into solution of PEO-b-PMA/Ca²⁺ complexes to activate carboxylic acid of PMA segments. Aqueous solution of cystamine as a cross-linker containing disulfide bonds was introduced into the dispersion of PEO-b-PMA/Ca²⁺ micelles and allowed to stir overnight at room temperature. The extent of degree of cross-linking was controlled by the ratio of amine functional groups of the cystamine to carboxylic acid groups of the copolymer. The 1.5 molar amounts of EDTA were added after completion of the reaction and followed by dialysis to remove Ca²⁺ ions and byproducts of the cross-linking reaction.

Characterization of Cross-linked Micelles with Biodegradable Ionic Cores

The physicochemical characteristics of these systems (size, zeta potential and morphology) were evaluated as functions of pH, ionic strength and the targeted degree of cross-linking (%). Zeta-potential (ζ) and effective diameter of the particles were be determined by dynamic light scattering (DLS) using ZetaPlus Analyzer with multi-angle sizing option (Brookhaven Instrument Co.). The biodegradability and morphology of the cl-micelles were evaluated by DLS and atomic force microscopy (AFM) methods.

Drug loading and Release studies

DOX or CDDP was immobilized into the cl-micelles by mixing with aqueous dispersion of polymer micelles. Unbound drugs were removed by ultrafiltration using Amicon YM-30 centrifugal filter (MWCO 30 KDa.). UV-Vis spectroscopy and HPLC (Agilent 1200 HPLC system) techniques were used to determine DOX loading efficacy and drug release profiles. ICP-MS was used for CDDP assays. The release studies were conducted in phosphate buffered saline (PBS, pH 7.4, 0.14 M NaCl) by dialysis method (Membrane with 3,500 Da cut-off) with or without glutathione.

In vitro Cytotoxicity studies

Cytotoxicity of DOX or CDDP-loaded cl-micelles was assessed in A2780 human ovarian carcinoma cells by a standard MTT assay. Cells was incubated to various doses (0–200 μg/ml on DOX or CDDP basis) of free drug, cl-micelles alone, drug-loaded cl-micelles/ED and drug-loaded cl-micelles for 24 h at 37 °C, followed by washing with PBS, and maintaining in RPMI 1640 medium with 10% FBS for additional 72 h. 25 μl of MTT indicator dye (5 mg/ml) was added to each well and the cells was incubated for 2 h at 37 °C in the dark. 100 μl of 50% DMF-20% SDS solution was added to each well and kept for overnight at 37 °C. Absorption was measured at 570 nm in a microplate reader (SpectraMax M5, Molecular Devices Co., USA) and obtained values was expressed as a percentage of the control cells to which no drugs were added.

Results and Discussion

Synthesis of cl-micelles

Polymer micelles with cross-linked ionic cores were prepared by using BIC of PEO-b-PMA copolymer and divalent metal cations as templates^1–2. Biodegradable disulfide bonds were introduced into the micellar core using cystamine as a cross-linker. The general scheme for the synthesis of cl-micelles is presented in Figure 1.

Figure 1.

Scheme for the synthesis of polymer micelles with disulfide bonds.

The resulting cl-micelles represented hydrophilic nanospheres of core-shell morphology. The core comprised a network of the cross-linked polyanions (PMA), which was surrounded by the shell of hydrophilic PEO chains.

Physicochemical characterization of cl-micelles

The physicochemical properties of cl-micelles were strongly affected by pH and degree of cross-linking. The particle size and net negative charge of cl-micelles increased considerably with increasing of pH. cl-micelles with 20% of degree of cross- liking swelled from ca.86 nm to ca. 140 nm with increase of pH from 6 to 9. Increase of targeted degree of cross-linking led to decrease of the particle size of polymer micelles at any given pH value (Figure 2).

Figure 2.

Physicochemical characterizations of cl-micelles. (a) Effective diameter (D_eff) and ζ-potential of cl-micelles with 20% targeted degree of cross-linking as a function of pH. (b) D_eff of cl-micelles at various pH as a function of targeted degree of cross-linking.

Biodegradability of disulfide bonds in the cl-micelles were evaluated by dynamic light scattering (DLS) and Atomic Force Microscopy (AFM) studies. The cl-micelles with disulfide bonds were degradable in the presence of reducing agent, dithiothreitol (DTT). Degradation of disulfide bonds in the cl-micelles caused initial swelling of the micelles which led to ultimate structural disintegration with resultant decrease in the particle size of the micelles (data not shown). The size of cl-micelles did not change in the absence of DTT. The changes in the topology of the cl-micelles as a result their degradation was further studied by AFM. In the absence of DTT, cl-micelles appeared to be spherical particles with the number averaged height of 4.3 ± 0.03 nm and diameter of 38.5 ± 0.18 nm (Figure 3a). The incubation of the cl-micelles dispersions with DTT (25mM, 37°C) for 3 hours resulted in a dramatic change in the topology of cl-micelles (Figure 3b). The mixed population of tiny particles and fibrous materials were observed in the AFM image. Tapping mode AFM imaging clearly demonstrated that cl-micelles with disulfide linkages can be degraded in the presence of reducing agents.

Figure 3.

Tapping mode AFM images of cl-micelles with 70% of degree of cross-linking in air, (a) before and (b) after incubation with DTT (25mM) for 3h at 37°C. Scan size is 2 μm.

Incorporation and release of anti-cancer drug in cl-micelles

Anticancer drugs, DOX and CDDP, were successfully incorporated into the ionic cores of cl-micelles. Incorporation of DOX or CDDP into the cl-micelles led to a decrease of particle size and net negative charge of the micelles (Table 1). This provides evidence of the progressive neutralization of the PMA segments in the cross-linked ionic core due to the binding of DOX or CDDP.

Table 1.

Physicochemical properties of drug-loaded cl-micelles with biodegradable ionic cores.

Drug	Deff (nm)	PD	Zeta-potential	LE (%)
DOX	109.9 ± 2.2	0.071	−14.3 ± 2.5	72.9
CDDP	91.2 ± 1.7	0.120	−1.88 ± 1.9	56.0

^*PD: polydispersity, LE: loading efficacy

The highest loading efficiency (percentage of drug incorporated into the micelles for a given amount added to the mixture) of DOX (ca. 73%) or CDDP (ca. 56%) into cl-micelles was achieved at pH 7.0 or pH 9.0, respectively. The loading level of drug into the cl-micelles was 51.1 w/w% for DOX and 42.9 w/w% for CDDP. The release profiles were examined for the cl-micelles loaded with DOX or CDDP under redox conditions using equilibrium dialysis. Both types of cl-micelles displayed sustained release of drug at the physiological conditions as shown in Figure 4 and no burst release was observed. Incubation with 10mM glutathione resulted in a significant enhancement of dug release from cl-micelles (Figure 4). These results clearly demonstrated that degradation of cl-micelles could trigger the release of encapsulated drug in a reducing environment.

Figure 4.

Release profiles of DOX (a) and CDDP (b) from cl-micelles in PBS buffer (0.14 M NaCl, pH 7.4) at 37 °C. Degree of cross-linking is 20%. (■) without and (□) in the presence of glutathione 10mM.

In vitro cytotoxicity

Cytotoxicity of degradable and non-degradable DOX- and CDDP-loaded cl-micelles was assessed in human A2780 ovarian cancer cells. Both types of cl-micelles alone were not toxic at concentrations used for the treatment by DOX- or CDDP-loaded cl-micelles formulations. In contrast degradable DOX or CDDP-loaded cl-micelles displayed nearly five fold higher cytotoxic activity than non-degradable cl-micelles (Table 2). These data might indicate that glutathione-induced intracellular biodegradation of the cl-micelles potentiated the release and cytotoxic activity of anti-cancer drugs.

Table 2.

Cytotoxic effect of DOX or CDDP-loaded cl-micelles in A2780 ovarian carcinoma cells (n=8).

Sample	IC50 (μg/mL)a	Relative Indexb
DOX	0.024	1
Degradable cl-micelles	0.058	2.5
Non-degradable cl-micelles	0.381	16.2
CDDP	0.254	1
Degradable cl-micelles	1.082	4.3
Non-degradable cl-micelles	5.997	23.7

^aIC₅₀ represents the concentration of a drug for 50% inhibition in vitro.

^bRelative index represents the IC₅₀ ratio between a control and cl-micelles for comparison.

Conclusions

Polymer micelles with biodegradable cross-linked cores were prepared using cystamine as a cross-linker. The resulting cross-linked micelles represented hydrophilic nanospheres of core-shell morphology. The ionic cross-linked PMA core of the micelles imparted pH-responsive hydrogel-like behavior to these nanostructures and allowed for the encapsulation of doxorubicin and cisplatin with high loading capacity. The biodegradation of cross-linked micelles under intracellular redox conditions can trigger the release of incorporated drugs and ensure the removal of empty carrier after drug release. These novel biodegradable cross-linked micelles are expected to be attractive nanocarriers for delivery of anticancer drugs.