Fmoc-Conjugated PEG-Vitamin E₂ Micelles for Tumor-Targeted Delivery of Paclitaxel: Enhanced Drug-Carrier Interaction and Loading Capacity

Abstract

The purpose of this study is to develop an improved drug delivery system for enhanced paclitaxel (PTX) loading capacity and formulation stability based on PEG_5K-(vitamin E)₂ (PEG_5K-VE₂) system. PEG_5K-(fluorenylmethoxycarbonyl)-(vitamin E)₂ (PEG_5K-FVE₂) was synthesized using lysine as the scaffold. PTX-loaded PEG_5K-FVE₂ micelles were prepared and characterized. Fluorescence intensity of Fmoc in the micelles was measured as an indicator of drug-carrier interaction. Cytotoxicity of the micelle formulations was tested on various tumor cell lines. The therapeutic efficacy and toxicity of PTX-loaded micelles were investigated using a syngeneic mouse model of breast cancer (4T1.2). Our data suggest that the PEG_5K-FVE₂ micelles have a low CMC value of 4 μg/mL and small sizes (~60 nm). The PTX loading capacity of PEG_5K-FVE₂ micelles was much higher than that of PEG_5K-VE₂ micelles. The Fmoc/PTX physical interaction was clearly demonstrated by a fluorescence quenching assay. PTX-loaded PEG_5K-FVE₂ micelles exerted more potent cytotoxicity than free PTX or Taxol formulation in vitro. Finally, intravenous injection of PTX-loaded PEG_5K-FVE₂ micelles showed superior anticancer activity compared with PEG_5K-VE₂ formulation with minimal toxicity in a mouse model of breast cancer. In summary, incorporation of a drug-interactive motif (Fmoc) into PEG_5K-VE₂ micelles represents an effective strategy to improve the micelle formulation for the delivery of PTX.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9651-2) contains supplementary material, which is available to authorized users.

INTRODUCTION

Paclitaxel (PTX) is a potent anticancer agent acting through the stabilization of microtubules (1). However, the clinical benefit of PTX is limited by its poor water solubility. The widely-used clinical formulation Taxol® contains alcohol and Cremophor EL which can cause irritation and hyperactivity reactions (2). Therefore, substantial efforts have been made to develop alternative formulations to improve the solubility of PTX. Polymeric micelles have attracted significant attention due to their unique advantages for the intravenous delivery of paclitaxel and other hydrophobic drugs. The small size (10–100 nm) of micelles and hydrophilic corona shells of PEG are critical for the long blood circulation time and effective passive targeting to tumor tissues through enhanced permeability and retention (EPR) effect (3), leading to enhanced antitumor efficacy and decreased toxicity. Several micelle formulations of PTX have been developed and hold great promise for clinical application, such as NK105 (PTX-loaded polyethylene glycol-(l-aspartic acid) micelles) (4) and Genexol-PM (a PTX incorporated PEG-PLA micelle system) (5).

d-alpha-tocopheryl polyethylene glycol (PEG)-1000 succinate (TPGS) is a PEG derivative of vitamin E that has been used as an excipient in various drug formulations (6–9). TPGS has intrinsic P-gp inhibitory effect which helps to overcome multidrug resistance (10,11). TPGS is self-assembled into micelles in aqueous solution; however, the high critical micelle concentration (CMC) decreases the stability of micelles in physiological environment (12). We and others have synthesized a series of PEG-vitamin E conjugates that differ in PEG molecular weight (2 vs. 5 K) and the number of vitamin E molecules (1 vs. 2) (13,14). By increasing the PEG length and including one additional VE, the PEG_2K-VE₂ micelles achieved lower CMC value, higher loading capacity for doxorubicin, and enhanced anticancer effect compared with PEG_2K-VE and traditional TPGS (13). In addition, our study showed that PEG_5K-(vitamin E)₂ (PEG_5K-VE₂) was the most stable and effective micellar formulation of PTX compared with PEG_2K-VE, PEG_2K-VE₂, and PEG_5K-VE (14). The tumor growth inhibitory effect of PTX-loaded PEG_5K-VE₂ was significantly improved in comparison to Taxol formulation or other three PTX micellar formulations in vivo (14). Nevertheless, the drug loading capacity of PTX-loaded PEG_5K-VE₂ micelles was still inadequate, which warranted further improvement.

We have recently shown that incorporation of a drug-interactive domain into a lipid surfactant can improve both drug-loading capacity and formulation stability. This concept was experimentally tested when we were formulating JP4-039, a mitochondria-targeted antioxidant (15). We have synthesized a series of PEG-lipopeptides that have a PEG hydrophilic headgroup, one or several oleoyl chains, and amino acids derivatized with various functional motifs at the interfacial region. Among several functional groups tested, 9-fluorenylmethoxycarbonyl (α-Fmoc) was identified to be the most effective drug-interactive motif that not only enhances the JP4-039 loading capacity but also improves the stability of JP4-039-loaded emulsion and micellar formulations (15). The improved drug/carrier interaction can be attributed to the hydrophobic/hydrophobic interaction, hydrogen bonding, and π − π stacking between the Fmoc-containing carrier and JP4-039 (15).

We hypothesize that inclusion of Fmoc moiety shall similarly improve the drug loading capacity of PEG_5K-VE₂ micelles for PTX. In the current work, we synthesized a novel amphiphilic polymer, PEG_5K-(fluorenylmethoxycarbonyl)-(vitamin E)₂ (PEG_5K-FVE₂) by introducing an Fmoc motif into PEG_5K-VE₂ conjugate. To evaluate the ability of PEG_5K-FVE₂ micelles for delivery of PTX, the drug-loading capacity of those micelles was investigated. The interaction between Fmoc moiety and PTX molecule was analyzed by measuring the change of Fmoc fluorescence intensity at different drug to carrier ratios. Cytotoxicity of blank micelles and PTX-loaded micelles was studied against free PTX using various tumor cell lines. Finally, the therapeutic efficacy and toxicity of PTX-loaded micelles were investigated using a syngeneic mouse model of breast cancer (4T1.2).

MATERIALS AND METHODS

Materials

PTX (98%) was purchased from AK Scientific Inc. (CA, USA). Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Lonza (MD, USA). Methoxy-PEG 5000-OH, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Triton X-100, Dulbecco’s modified Eagle’s medium (DMEM), succinate anhydride, Boc-lys-(Boc)-OH, and Fmoc-lys-(Boc)-OH were all purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were from Invitrogen (NY, USA). d-alpha-tocopheryl was purchased from Tokyo Chemical Industry (OR, USA). N,N′-dicyclohexylcarbodiimide (DCC) was purchased from Alfa Aesar (MA, USA). 4-Dimethylaminopyridine (DMAP) was purchased from Calbiochem-Novabiochem Corporation (CA, USA). All solvents used in this study were HPLC grade.

Synthesis of PEG_5K-Fmoc-VE₂ (PEG_5K-FVE₂)

PEG_5K-FVE₂ was synthesized via solution-phase condensation reactions from MeO-PEG-OH with a molecular weight of 5,000 Da. Fmoc-lys-(Boc)-OH (4 eq.) was coupled onto the terminal -OH of PEG using DCC (4 eq.) and DMAP (0.2 eq.) as coupling reagents in DCM for overnight. Fmoc-lys-(Boc) PEG ester was precipitated and washed three times with cold ethanol and ether, respectively. Then, Boc groups were removed by the treatment with 50% trifluoroacetic acid in DCM, and the Fmoc-lysyl PEG ester was precipitated and washed three times with cold ethanol and ether, respectively. Boc-lys-(Boc)-OH (2 eq.) was coupled onto the terminal -NH₂ of Fmoc-lysyl PEG ester using DCC (2 eq.) and DMAP (0.1 eq.) as coupling reagents in DCM for overnight. The di-Boc PEG ester was precipitated and washed three times with cold ethanol and ether, respectively. Then, Boc groups were removed by the treatment with 50% trifluoroacetic acid in DCM, and the di-NH₂ PEG ester was precipitated and washed three times with cold ethanol and ether, respectively. The resulting white powder precipitate was dried under vacuum. Vitamin E succinate (4 eq.) was coupled to the deprotected amino groups of lysine with the assistance of DCC (4 eq.) and DMAP (0.2 eq.). The resulting PEG_5K-FVE₂ was precipitated and washed three times with cold ethanol and ether, respectively. The final product was subsequently dialyzed against water and lyophilized to yield white powder.

Preparation and Characterization of PEG_5K-FVE₂ Micelles

Drug-free or PTX-loaded PEG_5K-FVE₂ micelles were prepared according to published procedures (14). Briefly, PTX solution (10 mM in chloroform) was mixed with PEG_5K-FVE₂ (10 mM in chloroform) at various molar ratios. The chloroform was removed by nitrogen flow and a thin dry film of drug/carrier mixture was formed. High vacuum was then applied to remove any traces of remaining solvent. DPBS was added into each vial to hydrate the film. The samples were then probe-sonicated for 2 min at 3 W. The blank micelles were similarly prepared except that PTX was not added.

The size of micelles was measured by dynamic light scattering (DLS) method using a Malvern Zetasizer (Nano ZS; Malvern instruments Ltd., Worcestershire, UK). The PTX concentration was kept at 1 mg/mL for DLS measurements. To measure the PTX loading efficiency, unincorporated free PTX was removed by filtering through a syringe filter with pore size of 0.22 μm (16). The amount of PTX loaded into micelles was analyzed by HPLC according to a published method (16). The drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated according to the following formula (16):

$$\begin{array}{l}\mathrm{DLC}\left(\%\right)=\left[\mathrm{weight}\mathrm{of}\mathrm{drug}\mathrm{used}/\left(\mathrm{weight}\mathrm{of}\mathrm{polymer}+\mathrm{drug}\mathrm{used}\right)\right]\times 100\%\hfill \\ \mathrm{DLE}\left(\%\right)=\left(\mathrm{weight}\mathrm{of}\mathrm{loaded}\mathrm{drug}/\mathrm{weight}\mathrm{of}\mathrm{input}\mathrm{drug}\right)\times 100\%\hfill \end{array}$$

CMC Measurement

The CMC of PEG_5K-FVE₂ was determined by employing pyrene as a fluorescence probe as described previously (16). Briefly, aliquots of 50 μL pyrene solution (4.8 × 10⁻⁶ M) were mixed with various amount of PEG_5K-FVE₂ in chloroform. The chloroform was then removed by nitrogen flow and high vacuum to form a thin film in the vials. Each sample was hydrated with 400 μL DPBS to give a final pyrene concentration of 6 × 10⁻⁷ M with PEG_5K-FVE₂ concentrations ranging from 6 × 10⁻⁵ to 0.25 mg/mL. The samples were kept shaking at 37°C for 24 h to reach equilibrium, followed by fluorescence measurement at the wavelength of em/ex = 334 nm/390 nm using Synergy H1 Hybrid Multi-Mode Microplate Reader (Winooski, VT). The fluorescence intensity was plotted against PEG_5K-FVE₂ concentration. The CMC was determined from the threshold concentration, where the fluorescence intensity begins to increase dramatically.

Fluorescence Measurement of Blank and PTX-Loaded Micelles

Micelle formulations with or without PTX were prepared in 500 μL of water as described (17). Freshly prepared micelles were added to 96-well plate (100 μL/well), and the fluorescence intensity was measured immediately with a Synergy H1 Hybrid reader (BioTek). The emission spectra were recorded ranging from 300 to 400 nm with a fixed excitation at 270 nm.

Hemolytic Effect of Micelles

Rat blood was collected via cardiac puncture with heparin as the anticoagulant. The blood sample was centrifuged at 1,500 rpm for 10 min at 4°C to collect the red blood cells (RBCs). The RBCs were washed with 30-mL ice-cold DPBS for three times and diluted to 2% w/v with ice-cold DPBS. One milliliter of diluted RBC suspension was mixed with blank PEG_5K-VE₂ or PEG_5K-FVE₂ micelles to obtain the final polymer concentrations of 0.2 or 1.0 mg/mL. The mixture was incubated at 37°C with constant shaking for 4 h and then centrifuged at 1,500 rpm for 10 min at 4°C. One hundred microliter of supernatant from each sample was transferred into a 96-well plate. The absorbance at 540 nm was measured using a PerkinElmer LS 50B luminescence spectrometer (Waltham, MA) to determine the release of hemoglobin from RBCs. Triton X-100 (2%) and DPBS were also incubated with RBCs as positive and negative controls, respectively. The percentage of hemoglobin release was calculated as (OD_sample − OD_{negative control})/(OD_{positive control} − OD_{negative control}) × 100% (16).

Cell Culture

Huh-7 is a human hepatocellular carcinoma cell line and Hepa 1–6 is a mouse hepatoma cell line. 4T1.2 is a mouse metastatic breast cancer cell line and MCF-7 is a human breast cancer cell line. All cell lines were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO₂.

In Vitro Cytotoxicity Study

The PTX-loaded PEG_5K-FVE₂ micelles were examined for their cytotoxicity against four cancer cell lines using PTX-loaded PEG_5K-VE₂ micelles, Taxol® formulation, and PTX dissolved in DMSO as control groups. The 4T1.2 cells were seeded in 96-well plates at a density of 1,000 cells per well. After incubation for 24 h, the cells were treated with various formulations at the indicated PTX concentrations for 72 h, followed by MTT assay as described previously (16). Briefly, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in DPBS (5 mg/mL) was added to each well and the cells were further incubated for 4 h. The medium was then removed and 150 μL DMSO was added into each well to solubilize the MTT formazan. The absorbance at 570 nm was detected by PerkinElmer LS 50B luminescence spectrometer (Waltham, MA). Data were shown as the percentages of cell viability calculated from triplicate wells using the formula: [(OD_treat − OD_blank)/(OD_untreat − OD_blank) × 100%]. The cytotoxicity with Huh-7, Hepa 1–6, and MCF-7 cells was similarly performed except that the cells were seeded at a density of 5,000 cells per well.

Animals

Female BALB/c mice, 10–12 weeks and male Sprague–Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). All animals were housed under pathogen-free conditions according to AAALAC guidelines. All animal-related experiments were performed in full compliance with institutional guidelines and approved by the animal use and care administrative advisory committee at the University of Pittsburgh.

Tissue Distribution of PTX-Loaded PEG_5K-FVE₂ Micelles

4T1.2 cells (2 × 10⁵ in 200 μL PBS) were inoculated s.c. at the right flank of female BALB/c mice. Once the tumor volume reached approximately 400–600 mm³, 15 mice were divided into 3 groups randomly and treated via tail vein injection with PTX-loaded formulations including PEG_5K-VE₂, PEG_5K-FVE₂ micelles, and Taxol at a PTX dose of 10 mg/kg. Mice were sacrificed 24 h after injection. The liver, lung, and tumor tissues were collected, weighed, and stored at −80°C.

Analysis of Tissue Samples

Mouse tissue was placed in glass tubes and homogenized with 2 mL solvent (acetonitrile to H₂O = 1:1, v/v). The samples were centrifuged at 4°C, 3,500 rpm for 15 min, and the supernatant was transferred to clean tubes. The tissue homogenates were dried by evaporation under nitrogen flow, then the residue was dissolved in 200 μL solvent (MeOH to H₂O = 1:1, v/v) and centrifuged at 4°C, 14,500 rpm for 6 min. The supernatant was mixed with equal volume of MeOH and centrifuged again at 4°C, 14,500 rpm for 6 min. Then, 20 μL of the clear supernatant was injected into HPLC system (16).

In Vivo Therapeutic Study

A syngeneic murine breast cancer model was used to evaluate the therapeutic efficacy of PTX-loaded PEG_5K-FVE₂ micelles. 4T1.2 cells (2 × 10⁵ in 200 μL PBS) were inoculated s.c. at the right flank of female BALB/c mice. Treatments were initiated when tumors in the mice reached a volume around 50 mm³ and this day was designated as day 0. On day 0, mice were randomly divided into five groups (n = 5) and treated with tail vein injection of saline (control), blank PEG_5K-FVE₂ micelles, PTX-loaded PEG_5K-VE₂ micelles (PEG_5K-VE₂ to PTX = 5:1, mol/mol), PTX-loaded PEG_5K-FVE₂ micelles (PEG_5K-FVE₂ to PTX = 5:1, mol/mol), and Taxol® (all at 10 mg PTX/kg), respectively. Blank PEG_5K-FVE₂ micelles were given at the equivalent dosage of the carrier in the group of PTX-loaded micelles. The treatment was performed three times a week for 2 weeks. Tumor size was measured with digital caliper three times a week. Tumor volume was calculated by the formula: (L × W²)/2, where L is the longest and W is the shortest in tumor diameters (mm). Mice were sacrificed at the end of experiment. To compare between groups, relative tumor volume (RTV) was calculated at each measurement time point (where RTV equals the tumor volume at a given time point divided by the tumor volume prior to the first treatment). Mice were euthanized when tumor volume reached 2,000 mm³. To monitor potential toxicity, the body weight of each mouse was measured three times a week. In addition, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were examined at the completion of the study.

Statistical Analysis

Statistical analysis was performed by Student’s t test for two groups, and one-way ANOVA for multiple groups, followed by Newman-Keuls test if P < 0.05. All results were reported as the mean ± standard deviation (SD) unless otherwise indicated. A value of P < 0.05 was considered statistically significant.

RESULTS

Synthesis and Biophysical Characterization of PEG_5K-FVE₂

PEG_5K-FVE₂ polymer was synthesized by conjugating PEG_5K with vitamin E succinate using dendritic oligo-lysine as the scaffold. Fmoc-lys-(Boc) and Boc-lys-(Boc) were sequentially linked to PEG_5K by condensation reaction, followed by conjugation with succinate VE. The synthetic scheme is shown in Fig. 1. The structure of PEG_5K-FVE₂ was confirmed by ¹H NMR (Fig. S1A) and MALDI-TOF (Fig. S1B). In Fig. S1A, the peaks between 7.2 and 8.5 ppm were assigned to the Fmoc group. The peak at 3.3 ppm corresponded to the –OCH₃ on the end of mPEG, the intense peaks at 3.5 ppm could be assigned to the methane protons of the polyethylene glycol. The proton peaks below 3.0 ppm were assigned to the vitamin E succinate. MALDI-TOF indicated that the molecular weight of PEG_5K-FVE₂ was around 6,500 (Fig. S1B), which is in agreement with the calculated molecular weight of 6,502.

Fig. 1.

Synthesis scheme of PEG_5K-FVE₂ conjugate

The sizes of PTX-loaded PEG_5K-FVE₂ micelles at different carrier to drug molar ratios were determined by DLS analysis. The sizes of blank and drug-loaded micelles were both around 60 nm (Table I). PTX could be loaded into PEG_5K-FVE₂ micelles at a carrier to drug molar ratio as low as 0.5:1. This is in contrast to PEG_5K-VE₂ micelles for which a minimal carrier to drug molar ratio of 2.5:1 was required to solubilize PTX (14). In addition, the drug loading efficiency as determined by HPLC was also improved by increasing the carrier to drug ratios. More than 95% of the input PTX was incorporated into the micelles at a carrier to drug molar ratio of 1:1 and above (Table I).

Table I.

Comparison of PTX-loaded PEG_5K-VE₂ and PEG_5K-FVE₂ Micelles (n = 3)

Micelle	Molar ratioa	Size (nm)b	PDIc	DLC (%)d	DLE (%)e
fPEG5K-VE2	–	18.8 ± 0.12	0.09	–	–
PEG5K-FVE2	–	60.12 ± 2.99	0.208	–	–
fPEG5K-VE2:PTX	2.5:1	20.1 ± 0.72	0.05	5.4	86.4
	5:1	19.7 ± 0.38	0.04	2.8	88.4
PEG5K-FVE2:PTX	0.5:1	55.32 ± 1.84	0.256	20.8	87.0
	1:1	61.60 ± 3.55	0.213	11.6	96.0
	2.5:1	68.64 ± 2.90	0.224	5.0	95.4
	5:1	70.06 ± 6.84	0.226	2.6	98.9

^aThe PTX concentration was 1 mg/mL in all the drug-loaded samples above

^bData represents the mean ± standard deviation (n = 3)

^cPDI: polydispersity index

^dDLC: drug loading capacity

^eDLE: drug loading efficiency

^fPEG_5K-VE₂: data from our previous publication (14)

The CMC of PEG_5K-FVE₂ polymer was measured with pyrene as the fluorescent probe. A series of pyrene-loaded PEG_5K-FVE₂ micelles were prepared with a fixed concentration of pyrene and different concentrations of polymer. When the concentration of a polymer reaches its CMC, the micellization is marked by an abrupt increase in fluorescence intensity of pyrene at the wavelength of 390 nm (18). Based on this principle, the fluorescence intensity was recorded and plotted against the logarithmic concentration of the PEG_5K-FVE₂ polymer, and the CMC value was determined. Figure 2 shows that the CMC of PEG_5K-FVE₂ micelles was 4 μg/mL, which was slightly higher than that of PEG_5K-VE₂ (1.7 μg/mL) (14), but still significantly lower than that of TPGS (0.2 mg/mL) (12).

Fig. 2.

CMC determination of the PEG_5K-FVE₂ micelles using pyrene as a hydrophobic fluorescence probe. The fluorescence intensity of pyrene was measured (Ex/Em = 334 nm/390 nm) and plotted against the logarithmic concentration of PEG_5K-FVE₂ micelles. The concentration of pyrene was kept at 6 × 10⁻⁷ mol/L. Values reported are the means ± SD, n = 3

Fluorescence Quenching Assay

The improved PTX loading capacity of PEG_5K-FVE₂ over PEG_5K-VE₂ suggests a role of Fmoc/PTX interaction in the formation of PTX-loaded PEG_5K-FVE₂ micelles. To test this hypothesis, we studied the Fmoc/PTX interaction via fluorescence quenching assay. We chose an excitation wavelength of 270 nm and examined the emission spectra of PEG_5K-FVE₂, PEG_5K-VE₂, and free PTX ranging from 300 to 400 nm. As shown in Fig. 3a, there is a distinct fluorescence peak at the emission wavelength of 305 nm for PEG_5K-FVE₂. This fluorescence appeared to be highly specific for Fmoc because minimal fluorescence intensity was detected for either PEG_5K-VE₂ or free PTX (Fig. 3a). We then prepared several PTX-loaded PEG_5K-FVE₂ micelles with constant PEG_5K-FVE₂ concentration but varying amounts of PTX, and similarly measured the fluorescence intensity. As shown in Fig. 3b, the fluorescence intensity of Fmoc group at 305 nm was dramatically reduced with increased amount of PTX. We also conducted a similar experiment with several different mixed micelles that had a fixed concentration of PTX (0 or 0.1 mg/mL) but varying amounts of PEG_5K-FVE₂. As shown in Fig. 3c, at each concentration of PEG_5K-FVE₂, the Fmoc fluorescence intensity at 305 nm was dramatically reduced in the presence of PTX. Figure 3d summarizes the changes of fluorescence intensity at 305 nm from the above fluorescence studies. It is apparent that the carrier to drug molar ratio affected the degree of reduction in fluorescence, which became more dramatic with increased amount of PTX.

Fig. 3.

Change of Fmoc fluorescence intensity in PEG_5K-FVE₂ loaded with various amount of PTX. The fluorescence emission spectra at 300–400 nm were recorded with a fixed excitation wavelength of 270 nm (a–c). b Fluorescence spectra at different drug to carrier molar ratios with fixed concentration of PEG_5K-FVE₂ at 3.81 mg/mL. c Fluorescence spectra at different drug to carrier molar ratios with fixed concentration of PTX at 0.1 mg/mL. d Summary of changes in the fluorescence intensity of various formulations in c at 305 nm

Hemolytic Effect of Micelles

The PEG_5K-FVE₂ polymer is amphiphilic and could solubilize lipids or insert into phospholipid membranes to induce membrane destabilization (19). Detrimental interaction between the polymeric materials and blood constituents such as red blood cells must be avoided for intravenous drug delivery. Therefore, the hemolytic effect of PEG_5K-FVE₂ micelles was evaluated as an indicator of the hematocompatibility. Polyethylenimine (PEI), a cationic polymer with potent cell surface activity, was included as the positive control. As shown in Fig. 4, incubation of RBC with PEI caused significant hemolysis in a dose-dependent manner. In contrast, PEG_5K-FVE₂ caused less than 5% hemolysis at both 0.2 and 1 mg/mL, which is considered as nonhemolytic according to the Guiding Principles of Hemolysis Test [H]GPT4-1 (20). These data suggest that the PEG_5K-FVE₂ polymer is hemocompatible and suitable for intravenous administration.

Fig. 4.

Hemolytic activity of PEG_5K-FVE₂ compared to PEG_5K-VE₂ and PEI. The polymers (0.2 or 1 mg/mL) were incubated with rat red blood cells (RBCs) for 4 h at 37°C in an incubator shaker. The degree of RBCs lysis was determined by measuring the absorbance of solution at 540 nm after centrifuge. 2% Triton X-100 and DPBS were used as positive and negative control, respectively. Values are presented as means ± SD, n = 3

In Vitro Cytotoxicity of PTX-loaded Micelles

The cytotoxicity of PTX-loaded PEG_5K-FVE₂ micelles was investigated in breast cancer cell lines such as 4T1.2, MCF-7, and liver cancer cell lines including Hepa 1–6 and Huh-7 cells (Fig. 5a–d). PTX-loaded PEG_5K-VE₂ micelles, free PTX dissolved with DMSO, and Taxol were included as control formulations. PTX-loaded PEG_5K-FVE₂ and PEG_5K-VE₂ micelles showed comparable tumor cell killing effect in all of the cell lines tested (Fig. 5a–d). In 4T1.2 cancer cells, both PTX-loaded PEG_5K-VE₂ and PEG_5K-FVE₂ micellar formulations were more cytotoxic than free PTX, but less active than Taxol formulation (Fig. 5a). However, the two PTX micellar formulations showed higher level of cytotoxicity than both Taxol and free PTX towards MCF-7 cells (Fig. 5b), Hepa 1–6 cells (Fig. 5c), and Huh-7 cells (Fig. 5d). The IC₅₀ of various formulations in the four tumor cell lines tested were summarized in Table S1. Carrier alone (PEG_5K-FVE₂ or PEG_5K-VE₂) showed minimal cytotoxicity at the concentrations used for PTX-loaded micelles (Fig. S2).

Fig. 5.

In vitro cytotoxicity of free PTX, PTX-loaded PEG_5K-FVE₂ micelles, PTX-loaded PEG_5K-VE₂ micelles, and Taxol formulation against several cancer cell lines including 4T1.2 (a), MCF-7 (b), Hepa 1–6 (c), and Huh-7 (d). Cells were treated with various formulations for 72 h followed by MTT assay. Data are reported as mean ± SD, n = 3

Tissue Distribution of PTX-loaded Micelles

Female BALB/c mice bearing 4T1.2 breast tumor were used for tissue distribution study. PTX-loaded PEG_5K-VE₂ micelles, PEG_5K-FVE₂ micelles, and Taxol formulation were injected at the same PTX dose of 10 mg/kg. At 24-h postinjection, liver, lung, and tumors were excised for the analysis of drug concentration. As shown in Fig. 6, PTX concentration in the tumor of mice treated with PEG_5K-FVE₂/PTX micelles was significantly higher than that of PEG_5K-VE₂/PTX and Taxol groups (Fig. 6).

Fig. 6.

Tissue distribution of PTX 24-h postinjection. PTX-loaded PEG_5K-VE₂ and PEG_5K-FVE₂ micelles were injected into female BALB/c mice bearing 4T1.2 breast tumor at the dose of 10 mg PTX/kg, respectively. Taxol was also injected as a control. Values are presented as means ± SD, n = 5

In Vivo Therapeutic Study

The in vivo tumor growth inhibitory effect of PTX-loaded PEG_5K-FVE₂ micelles were investigated in a syngeneic murine breast cancer model (4T1.2). As shown in Fig. 7a, blank PEG_5K-FVE₂ micelles showed minimal inhibitory effect on tumor growth compared with the control group treated with saline. Taxol exhibited moderate effect in inhibiting the tumor growth at the PTX dose of 10 mg/kg. In contrast, at the same PTX dose, a significant improvement in tumor growth inhibition was achieved in the mice treated with PTX-loaded PEG_5K-VE₂ micelles. Furthermore, the antitumor effect of PTX-loaded PEG_5K-FVE₂ micelles was even higher than that of PTX-loaded PEG_5K-VE₂ micelles.

Fig. 7.

a Enhanced antitumor activity of PTX formulated in PEG_5K-FVE₂ micelles in vivo. Balb/c mice were inoculated s.c. with 4T1.2 cells (2 × 10⁵ cells/mouse). After the tumor volume reach 50 mm³, the mice received various treatments via tail vein injection three times a week for 2 weeks as indicated by arrows in the graph. Tumor volume was measured and plotted as relative tumor volume. b Change of body weight in mice receiving different treatments. The ALT (c) and AST (d) enzyme activity in serum was measured at the conclusion of experiment. Data are presented as mean ± SD. *p < 0.05, PTX-loaded PEG_5K-FVE₂ micelles versus PTX-loaded PEG_5K-VE₂ micelles. n = 5

The toxicity of all of the formulations was evaluated by following their effects on body weight and serum levels of AST and ALT. No significant changes in body weight occurred in all of the groups (Fig. 7b). In addition, serum levels of AST and ALT in the mice treated with PTX-loaded PEG_5K-FVE₂ micelles were comparable to those in saline-treated group (Fig. 7c, d), suggesting the negligible toxicity of PTX-loaded PEG_5K-FVE₂in vivo.

DISCUSSION

We have previously shown that inclusion of drug-interactive Fmoc moiety into a lipopeptide surfactant can dramatically enhance its ability for loading JP4-039, a poorly water-soluble antioxidant (15). In this study, we applied this concept in the optimization of PEG_5K-VE₂ polymeric material and synthesized a novel compound PEG_5K-FVE₂. Our data showed that PEG_5K-FVE₂ is more effective for the loading of PTX compared with PEG_5K-VE₂.

Drug solubility in amphiphilic di-block copolymer micelles is affected by the compatibility between the drug and the micelle core (21). Accumulating data have demonstrated that the drug loading capacity and stability of micelles can be greatly improved by tailoring the inner core segment of micelles for stronger drug-carrier interaction. Besides hydrophobic interaction, the compatibility between drug and carrier was shown to be improved by various additional noncovalent bonding. For example, hydrogen-bonding of micelles with doxorubicin significantly enhanced their loading capacity and stability (22, 23). In addition, acid–base interaction between hydrophobic segments of micelles and guest molecules containing carboxylic acid groups also dramatically improved the loading of indomethacin and ibuprofen (24). Furthermore, metal-ligand coordination facilitated the encapsulation of cisplatin into polymeric micelles (25, 26). In particular, Park’s group showed that inclusion of “hydrotropic motifs” into the hydrophobic domain of polymeric micelles represented a versatile strategy to enhance the compatibility of carrier with poorly water-soluble drugs (27, 28). Similarly, inclusion of aromatic end groups into the hydrophobic chain of diblock copolymers improved the stable incorporation of camptothecin (29) and PTX (15, 30). Data from this study and another recent study (17) suggest that incorporation of a drug-interactive motif (Fmoc) at the interfacial region represents an effective strategy in improving the performance of lipid-core micellar systems in loading hydrophobic drugs such as PTX.

Various mechanisms are likely to be involved in the carrier/drug interactions of PTX-loaded PEG_5K-FVE₂ micelles, such as hydrophobic interaction with PTX, hydrogen bonding, and the π–π stacking effect (14). The enhanced PTX loading capacity was most likely due to excellent Fmoc-PTX compatibility, which results from the rigid aromatic structure of Fmoc moieties that may interact with those in PTX. This hypothesis was further supported by the change in Fmoc fluorescence intensity in the presence of PTX, which indicated the close proximity between Fmoc and PTX. The decrease of Fmoc fluorescence intensity likely results from the fluorescence quenching caused by intermolecular π–π interactions (31). This information not only supports a role of Fmoc in the improved PTX loading capacity of PEG_5K-FVE₂ micellar system, but also sheds new light on the design of novel polymeric nanocarriers for the delivery of other aromatic structure-containing drugs such as doxorubicin.

MTT assay showed that the cytotoxicity of PTX-loaded PEG_5K-FVE₂ and PEG_5K-VE₂ micelles was more potent than that of free PTX solubilized in DMSO in all the cell lines tested. In Hepa 1–6 and Huh-7 cells, the cell killing effect of PTX-loaded PEG_5K-FVE₂ and PEG_5K-VE₂ micelles was even higher than that of Taxol formulation at the same PTX concentrations. This might be attributed to the increased cellular uptake of PTX into the cells due to the well-known p-gp inhibitory effect of vitamin E derivatives (10, 14). The fact that the cytotoxicity of PTX-loaded PEG_5K-FVE₂ micelles was similar or slightly higher than that of PTX-loaded PEG_5K-VE₂ micelles suggests that the PEG_5K-FVE₂ retained the function of PEG_5K-VE₂ in enhancing the cellular uptake of drug-loaded micelles.

A highly metastatic murine breast cancer cell line 4T1.2 was selected to investigate the in vivo therapeutic efficacy of PTX-loaded PEG_5K-FVE₂ micelles. PTX-loaded PEG_5K-FVE₂ micelles demonstrated significantly higher tumor inhibitory effect compared with both Taxol and PEG_5K-VE₂ formulations at the same PTX concentration. The superior antitumor efficacy of PTX-loaded PEG_5K-FVE₂ micelles compared with PEG_5K-VE₂ micelles can be attributed to the enhanced tumor accumulation of PTX (Fig. 6). To better understand the mechanism for the enhanced tumor uptake of PEG_5K-FVE₂ micelles, we observed the morphology of blank and PTX-loaded micelles with transmission electron microscopy (Fig. S3). In contrast to the PEG_5K-VE₂ micelles that form typical spherical structures, PEG_5K-FVE₂ micelles showed predominantly elongated, worm-like morphology (Fig. S3). This might be attributed to the strong intermolecular interaction among the PEG_5K-FVE₂ molecules mediated by Fmoc moieties. Fmoc is known to promote parallel interactions of individual short peptides carrying the same group, which often leads to the formation of elongated nanoassemblies (15). It has been reported that drug carriers with worm-like shape, also known as filomicelles, show longer circulation time compared with spherical nanoparticles composed of the similar materials, leading to improved drug accumulation in tumors (32). More studies are ongoing in our lab to better understand the underlying mechanism for the improved performance of PEG_5K-FVE₂ micelles.

Besides measuring the mouse body weight as well as the serum levels of AST and ALT, we have observed that mice treated with Taxol frequently showed transient apathy (33) and decreased overall activity over 10-min postinjection. This is likely caused by Cremophor EL and ethanol in the Taxol formulation. In contrast, there was no noticeable change in activity of mice after injection of PTX-loaded PEG_5K-FVE₂ micelles, suggesting the minimal toxicity of PTX-loaded PEG_5K-FVE₂in vivo.

CONCLUSIONS

In summary, we have validated and extended our previous study on the significance of introducing a drug-interactive motif at the interfacial region of conventional micellar system. With an improved TPGS-based micellar carrier, PEG_5K-FVE₂, our data clearly showed that incorporation of an Fmoc drug-interactive motif not only enhanced the drug loading capacity of PTX, but also significantly improved the tumor accumulation and therapeutic efficacy. These effects are likely attributed to the high compatibility and interaction between PTX and Fmoc moiety. Our strategy may represent an effective and general approach to improve the performance of various delivery systems including micelles.

Abbreviations

PTX

Paclitaxel

PEG_5K-VE₂

PEG_5K-(vitamin E)₂

PEG_5K-FVE₂

PEG_5K-(fluorenylmethoxycarbonyl)-(vitamin E)₂

EPR

Enhanced permeability and retention

TPGS

d-alpha-tocopheryl polyethylene glycol (PEG)-1000 succinate

CMC

Critical micelle concentration

DLS

Dynamic light scattering

DLC

Drug loading capacity

DLE

Drug loading efficiency

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

AST

Aspartate aminotransferase

ALT

Alanine aminotransferase

PEI

Polyethylenimine