In this invention, a multi-component micelle was prepared from graft and diblock copolymers; the differences between the CMCs of the copolymers are used to control the particle size. Additionally, the mixed micelle in this structure can be extended for many applications by manipulating and carefully designing each component. One such application is as an anticancer drug carrier. Intracellular drug delivery is one of the important routes for being used in cancer therapy. This pathway enhances the cytotoxicity of drugs toward targeted cells and minimizes the side effects on normal tissue. The mechanisms for inducing the release of drugs from carriers after the uptake by cells involve lysosomal enzymes and a change in intracellular pH to deform the carriers. Many materials have been investigated and synthesized to achieve this pathway before the present invention. However, some of them, possessing hydrophobic groups or highly electronic charges, may be recognized by mononuclear phagocyte systems (MPS), cannot accumulate easily in tumor regions, and so are not suitable for use even in intravenous injection, far less intracellular drug delivery. Therefore, the hydrophilic segment extended on the surface of particle is necessary.
In one of the preferred embodiments of the present invention, a novel mixed micelle with a multi-functional core and shell was successfully prepared from an environmentally-sensitive graft copolymer, poly(N-isopropyl acrylamide-co-methacryl acid)-g-poly(D,L-lactide) (P(NIPAAm-co-MAAc)-g-PLA) and two diblock copolymers, methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PLA) and poly (2-ethyl-2-oxazoline)-b-poly(D, L-lactide) (PEOz-PLA). This nano-structure completely screens highly negative charges of the graft copolymer and exhibits multi-functions because it has a specialized core-shell structure. An example of this micelle structure in intracellular drug delivery demonstrated a strong relationship between drug release and the functionality of the mixed micelle. Additionally, the efficiency of screening feature also displayed in the cytotoxicities; mixed micelles exhibited higher drug activity and lower material cytotoxicity than micelles from P(NIPAAm-co-MAAc)-g-PLA ([NIPAAm]/[MAAc]/[PLA]=84:5.9:2.5 mol/mol). This embodiment not only presents a new micelle structure generated using a graft-diblock copolymer system, but also elucidates concepts on which the preparation of a multi-functional micelle from a graft copolymer with a single or many diblock copolymers can be based for applications in drug delivery.
In another preferred embodiments of the present invention, multifunctional micelles for cancer cell targeting, distribution imaging, and anticancer drug delivery were prepared from an environmentally-sensitive graft copolymer, P(NIPAAm-co-MAAc)-g-PLA, a diblock copolymer, mPEG-PLA and two functionalized diblock copolymers, galactosamine-PEG-PLA (Gal-PEG-PLA) and fluorescein isothiocyanate-PEG-PLA (FITC-PEG-PLA). Multifunctional micelles target specific tumors by an asialoglycoprotein (HepG2 cells)-Gal (multifunctional micelle) receptor-mediated tumor targeting mechanism. The intracellular pH changes then induce structural deformation of the P(NIPAAm-co-MAAc)-g-PLA graft copolymer inner core of multifunctional micelles and thereby increases HepG2 cell cytotoxicity by releasing doxorubicin (Dox). Confocal laser scanning microscopy (CLSM) reveals a clear distribution of multifunctional micelles. With careful design and sophisticated manipulation, polymeric micelles synthesized in the present invention can be widely used in cancer diagnosis, cancer targeting, and cancer therapy simultaneously.
The present invention will be better understood through the following examples which are merely for illustrative and not for the limitation of the scope of the present invention.
D,L-Lactide and methacrylic acid (MAAc) were purchased from Lancaster. Methyl p-toluenesulfonate (MeOTs), stannous octoate, 2-hydroxyethyl methacrylate (HEMA), pyrene and 2,2′-azobisisobutyronitrile (AIBN) were purchased from Aldrich. N-Isopropyl acrylamide (NIPAAm) and 2-ethyl-2-oxazoline were purchased from TCI. MPEG (weight-average molecular weight, Mw=5000 Da) was purchased from Sigma. D,L-Lactide was further purified by recrystallization from tetrahydrofuran (THF) twice before used. NIPAAm and AIBN were purified by recrystallization from hexane and acetone, respectively. MAAc and HEMA were purified by distillation under vacuum. 2-Ethyl-2-oxazoline and MeOTs were treated with CaH2 overnight and purified by distillation under vacuum. Other reagents were commercially available and were used as received.
First, PLA with an end-capping, methacrylated group (PLA-EMA) was synthesized by ring-opening polymerization. D,L-Lactide (4 g), HEMA (0.26 g) and toluene (5 mL) were added to a two-necked round-bottle flask with magnetic stirring. The flask was immersed in an oil bath and stirred at 130° C. under nitrogen. Stannous octoate (1 wt %) was then added to start the polymerization, which was continued for 16 h at 100° C. After polymerization, the product was terminated by adding 0.1 N methanolic KOH and then precipitated from diethyl ether twice. PLA-EMA with one end capped by a methacrylated group was obtained (Mn=2000). P(NIPAAm-co-MAAc)-g-PLA graft copolymer was synthesized by traditional free-radical copolymerization. PLA-EMA (0.35 g), NIPAAm (1.15 g), MAAc (0.16 g) and AIBN (0.023 g) were placed in a two-necked round-bottle flask with a magnetic stirring bar, and the mixture was dissolved in acetone (15 mL). The reaction was performed at 70° C. for 24 h under nitrogen. After polymerization, the product was purified twice by precipitation from diethyl ether and twice by precipitation from distilled water, to yield the final graft copolymer (P(NIPAAm-co-MAAc)-g-PLA ([NIPAAm]:[MAAc]:[PLA]=84:5.9:2.5 mol/mol (Graft I, G1).
MPEG-PLA diblock copolymer was synthesized by ring-opening polymerization. D,L-Lactide (1 g), mPEG (Mw=5000 Da) (10 g) and toluene (4 mL) were added to a two-necked round-bottle flask with a magnetic stirring bar. The mixture was heated in an oil bath and stirred at 130° C. under nitrogen. Stannous octoate (1 wt %) was then added to start the polymerization, which was continued for 16 h at 130° C. After polymerization, the product was terminated by adding 0.1 N methanolic KOH and recrystallizing from dichloromethane and diethyl ether cosolvent at −20° C. m PEG-PLA ([EG]:[LA]=113:7 mol/mol) was thus obtained (Block I, B1).
PEOz-PLA was prepared by the modification of procedures in the literature [G. H. Hsiue, C. Ch. Wang, C. L. Lo, C. H. Wang, J. P. Li, J. L. Yang, Int. J. Pharm. 2006, 317, 69], as follows. First, 2-ethyl-2-oxazoline (10 mL), the initiator methyl p-toluenesulfonate (0.232 mg) and acetonitrile (30 mL) were added to a two-necked round-bottle flask with a magnetic stirring bar. The flask was moved to an oil bath and the mixture was stirred at 100° C. under nitrogen for 30 h. After cooling to room temperature, the reaction was terminated by adding 0.1 N methanolic KOH and precipitating twice from diethyl ether twice to yield PEOz-OH. Then, PEOz-OH (2 g) and D,L-lactide (0.426 g) were polymerized using stannous octoate (1 wt. %) for 16 h at 130° C. under nitrogen. After polymerization, the product was terminated by adding 0.1 N methanolic KOH and precipitating twice from diethyl ether to yield PEOz-PLA ([EOz]:[LA]=52.5:9.7 mol/mol) (Block II, B2).
The chemical structure and polydispersity index of each copolymer prepared above were verified by 1H-NMR (AMX-500, Bruker) and GPC using dimethylformamide (DMF) as an elution solvent. The Mn of Graft was calculated by 1H-NMR (AMX-500, Bruker) using mPEG (Mn 2000) as a standard. Additionally, the critical micelle concentration (CMC) of each was identified using a fluorescence spectrometer with pyrene as a hydrophobic probe. The copolymer concentration varied from 0.0001 to 10 mg/mL. Fluorescence spectra were obtained using a fluorescence spectrophotometer (F-2500, Hitachi). The excitation wavelength for the emission spectra was 339 nm and excitation spectra were recorded at 390 nm. Table 1 summarizes those results.
Various compositional ratios of Graft I and Block I, with or without Block II, were dissolved together in DMSO to prepare a polymer solution. The polymer solution was then dialyzed against distilled water for 48 h at 20° C. using a cellulose membrane bag (with a molecular weight cut-off of 6000-8000, obtained from SpectrumLabs, Inc.). The distilled water was replaced every 3 h. After dialysis, micelle or mixed micelle solutions were collected and frozen using a freeze dryer system (Heto-Holten A/S, Denmark) to yield dried products.
Table 2 lists the concentrations of Graft I, Block I and Block II used in preparing two of the micelles shown in
Three copolymers of the mixed micelle G1B1B2 [G1:B1:B2=33.9:55.7:10.4 mol/mol] exhibited self-assembly, packing and association with hydrophobic PLA to form mixed micelles yielding a uniform particle size (182.3±1.5 nm) and a narrow distribution (polydispersity index, PDI=0.038±0.014), as determined by dynamic light scattering (DLS) from the sample in phosphate buffer saline (PBS) at a concentration of 0.1 mg/mL. The zeta-potential of the mixed micelle was measured by Doppler microelectrophoresis (Zetasizer 3000HS, Malvern) in PBS at a concentration of 0.1 mg/mL, to identify the effect of diblock copolymers on hiding efficiency. The micelle that was composed of Graft I was used as a comparative sample; the corresponding zeta-potential was measured to be −15.5±0.9 mV. The highly negative charge caused by the slight ionization of carboxyl acid groups of MAAc was screened by diblock copolymers in the mixed micelle. The zeta-potential of the mixed micelle was measured to be −7.8±1.3 mV, because the hydrophilic segments MPEG and PEOz were extended on the surface of the mixed micelles, hiding the carboxyl acid groups of MAAc. The most direct evidence of the mixed micelle structure is obtained by transmission electron microscopy (TEM; Hitachi H-600 microscope, accelerating voltage=100 kV), as shown in
Mixed micelles were dialyzed from Graft I with Block I (G1B1) and from Graft I and Block I with Block II (G1B1B2), to compare the effects of the compositions of the diblock copolymers on the preparation of mixed micelles. The three copolymers exhibited various CMCs: the CMC of Block I differed greatly from that of the Block II and Graft I (Table 1). When a fixed concentration of Graft I was treated with various molar ratios of Block I (CMCGraft I<<CMCBlock I), the average diameters of the G1B1 mixed micelles were smaller than those from single Graft I or single Block I, and remained constant at around 160 nm, as determined by DLS, as shown in
Poly(N-isopropylacrylamide) (PNIPAAm) is well known to be a water-soluble and hydrophilic polymer, that exhibits an extended chain conformation below the lower critical solution temperature (LCST) when it is in aqueous solution. PNIPAAm can also undergo a phase transition to an insoluble and hydrophobic aggregate above its LCST. Randomly copolymerizing a small proportion of the MAAc in PNIPAAm copolymers raises the LCST above 37° C. (i.e., body temperature) and causes the polymer to be sensitive to pH. P(NIPAAm-co-MAAc)s exhibits an extended chain in neutral surroundings. This is because the ionized MAAcs increase the hydrophilicity of P(NIPAAm-co-MAAc)s. In acidic surrounding, the copolymer aggregates and precipitates, owing to the fact that the de-ionized MAAcs decreases the hydrophilicity of P(NIPAAm-co-MAAc)s and reduces its LCST to 32° C. The pH-sensitive properties of MAAc and thermal-sensitive properties of PNIPAAm are correlated. Our previous study demonstrated that Graft I micelles exhibited a structural change because of aggregation and the collapse of the P(NIPAAm-co-MAAc) outer shells in response to the change of the temperature at low pH [C. L. Lo, K. M. Lin, G. H. Hsiue, J. Controlled Release 2005, 104, 477].
The inventors of the present invention also analyzed mixed micelles G1B1B2 [G1:B1:B2=33.9:55.7:10.4 mol/mol] before and after structural changes by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The chemical compositions of the surface layers of mixed micelles before and after the induced structural changes were determined from positive and negative TOF-SIMS spectra. The results from TOF-SIMS spectra indicate that the Graft I in the mixed micelles was exposed and closed the surface layer after the induced structural change. The mixed micelles after structural change were treated with uranyl acetate and monitored by TEM to yield further evidence of this finding. As shown in
In this example, a mixed micelle structure, composed of MPEG-PLA diblock copolymer and P(NIPAAm-co-MAAc)-g-PLA graft copolymer, was used to encapsulate a hydrophobic anticancer drug, free base doxorubicin (Dox), whose structure enables the encapsulated drug to remain in the core during circulation in the blood.
Doxorubicin (Dox)-loaded mixed micelle was also prepared by dialysis. The preparation procedures were similar to those of the mixed micelles prepared in Example 1. 20 mg of Dox-HCl was dissolved in 8 ml DMF and 2 ml DMSO. 2 mg of mPLA-b-PEG (Block I) and 20 mg of P(NIPAAm-co-MAAc)-g-PLA (Graft I) were dissolved in 8 ml DMF and 2 ml DMSO. The Dox-HCl solution was mixed with 0.3 ml of triethylamine to remove hydrochloride. Then, the free base Dox solution was added to the polymer solution and stirred at room temperature for 2 h. The mixed solution was dialyzed against water at 20° C. for 72 h. The distilled water was replaced every 3 h. After dialysis, the solution of micelles was collected and frozen using a freeze-drying system to yield dried micelles. Weighted amounts of the mixed micelles were dissolved in DMSO at room temperature for 12 h; they then underwent ultrafiltration (ultrafiltration membrane MWCO 1000, Millipore) and samples were removed and analysis to determine Dox content using a UV/Vis spectrometer at 485 nm by reference to a calibration curve of Dox in DMSO. Accordingly, the Dox content in the mixed micelles was determined. The drug content of mixed micelles was calculated using the formula: drug content (% w/w)=(total mass of Dox in mixed micelles)/(total mass of Dox in mixed micelles+total mass of polymer in mixed micelles)×100.
The mixed micelles incorporated with Dox (Dox-loaded mixed micelles) were formed with a uniform particle size of about 165 nm as shown in an AFM image.
Drug Release Assay. The release of Dox-loaded mixed micelles in pH 5.0 and pH 7.4 buffer solutions at 37° C. and 25° C., respectively, was examined. Dox released from mixed micelles was isolated from the mixed-micelle buffer solution (50 mg/L) by ultrafiltration (ultrafiltration membrane MWCO 10000, Millipore). The isolated solution was measured using a UV/Vis spectrometer at 485 nm in a time-course procedure.
Cytotoxicity Evaluation. The cytotoxicity of each sample was determined by measuring the inhibition of cell growth using a tetrazolium dye (MTT) assay. Dox-loaded mixed micelles and Dox-loaded Graft I micelles were washed twice with PBS to remove untrapped Dox before use. HeLa cells (5×103 cell/mL) harvested in a logarithmic growth phase were seeded on 96 wells in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37° C. After the HeLa cells had been incubated in a logarithmic growth phase, samples with various concentrations of Dox were added for 48 h of co-culturing. At the end of the experiment, the MTT assay was conducted and the percentage of cell viability was calculated. Additionally, material cytotoxicities were measured using HeLa cells (5×103 cell/mL). The experimental process was identical to that described above.
Internalization Evaluation. Accumulated Dox in HepG2 cells was localized using a Carl Zeiss LSM5 PASCAL confocal laser scanning microscope (CLSM). The HeLa cells were seeded on coverslides for 24 h and then treated with free Dox or Dox-loaded mixed micelles. Dox-loaded mixed micelles were washed with PBS twice to remove untrapped Dox before use. The concentration of Dox was ca. 10 μg/mL. After an interval, the cells were washed twice with PBS; then, the LysoTracker was added in a culture medium without FBS. After 30 min of incubation, the cells were washed with PBS and mounted on a slide with 4 wt % paraformaldehyde for CLSM observation. Fluorescence observation was carried out with a confocal microscope at 488 nm for excitation and an LP (long-pass) filter of 590 nm for Dox detection. Besides, LysoTracker observation was also carried out with a confocal microscope at 504 nm for excitation and an LP filter of 511 nm for detection.
UV/Vis spectrophotometry demonstrated that the drug content of the mixed micelle incorporating Dox was around 19%. The Dox released from mixed micelles was isolated from the micellar buffer solution using the ultrafiltration membrane.
HeLa cells (5×103 cell/ml) were used to study the cytotoxicity of free Dox and Dox-loaded mixed micelle by measuring the inhibition of cell growth using a tetrazolium dye (MTT) assay. Micelle that incorporated Dox from Graft I copolymer (Dox-loaded Graft I micelle) with a particle size of 176.2 nm and a drug content of about 17% was used for comparison. As shown in
Confocal laser scanning microscopy (CLSM) was used to observe the intracellular drug release of mixed micelles. Confocal images were taken to observe the time-dependent fluorescence intensity of LysoTracker and Dox after mixed micelles were incubated with HepG2 (human hepatocellular carcinoma) cells. The red fluorescence from Dox and the green fluorescence from LysoTracker were detected in the intracellular compartment. A LysoTracker molecule was an indicator while located in the acidic compartment. After both one hour and eight hours incubation of HepG2 with Dox, the fluorescence from free Dox was concentrated in the nucleus. Fluorescence from LysoTracker occurred in both the nucleus and cytoplasm because these are acidified by doxorubicin hydrochloride. After Dox-loaded mixed micelles had been exposed for 1 h, a small amount of Dox was released from the mixed micelles and observed in the cytoplasm, where the LysoTracker molecules were also located, indicating that the mixed micelles were taken up from the extracellular fluid into the cells by endocytosis, and the pH of the endosomal compartments were then changed, inducing the release of Dox. Eight hours later, Dox was released from mixed micelles, associated with a strong signal. Dox was localized not only in the cytoplasm but also accumulated in the nucleus. Similar results were obtained when Chinese hamster ovary cells (CHO-K1) were treated with Dox-loaded mixed micelles.
The Dox-loaded mixed micelles prepared in this example can rapidly be damaged to release Dox when the intracellular pH changes; it also has a hydrophilic outer shell that screens highly negative charges and increases its solubility.
Similar to the procedures in Example 1, Block III (mPEG5000-PLA1088, PDI=1.15, CMC=16 mg/L) and Block IV (mPEG5000-PLA1750, PDI=1.20, CMC=5.4 mg/L) copolymers were synthesized by ring-opening polymerization from methoxy poly(ethylene glycol) (mPEG, Mn 5000) and D,L-lactide using stannous octoate as a catalyst. These diblock copolymers have the same chemical nature, but differ in composition ratio.
Two-component mixed micelles composed of a graft copolymer (Graft I prepared in Example 1) and a diblock copolymer (Block I, Block III or Block IV) were employed to investigate the influence of chain length and CMC of the diblock copolymers on the morphology and structure of mixed micelles. First, a graft copolymer and a diblock copolymer were dissolved together in dimethylsulfoxide (DMSO)/dimethylformamide (DMF) (4/1 v/v) cosolvent to prepare a polymer solution. The DMF/DMSO solvent mixture was used because it produces the smallest mixed micelles. Graft copolymer concentration was fixed at 10 mg/mL. The molar ratio of the graft copolymer to the diblock copolymer was 1:9. Mixed micelles were then prepared by dialysis by using the procedures described in Example 1. The core-shell structure and particle size of three mixed micelles from a graft copolymer and a diblock copolymer (Block I, Block III or Block IV) were observed by transmission electron microscopy (TEM). TEM observation produced three results. (1) For all mixed micelles, the dark region of the graft copolymer is the inner core, and hydrophilic segments of mPEG extended outside the core. (2) The radius of the core region decreased as the chain length of PLA of diblock copolymer increased (PLA500>PLA1088>PLA1750). (3) Mixed micelle particle size increased as the chain length of PLA of diblock copolymer increased (PLA1750>PLA1088>PLA500). A short PLA length produces smaller mixed micelles.
A test for the stability of micelles in the presence of serum or serum albumins was conducted. In this test, mixed micelles (25 mol % of graft copolymer (Graft I) and 75 mol % of mPEG5000-PLA1750 (Block IV)) was chosen. The stability of mixed micelles was determined by dynamic light scattering (Zetasizer 3000HS, Malvern). Mixed micelles in PBS (2 mg/mL) were mixed with an equal volume of 4 wt % bovine serum albumin (BSA) dissolved in PBS. The mixture was incubated at 37° C. and determined by dynamic light scattering (DLS) at time interval, defined as ti. The CONTIN analytic method was used. The average diameter of micelles in PBS (1 mg/mL) before BSA treatment, to was also measured. The ratio of particle sizes was calculated as ti/t0. Results show that mixed micelles were stable after 72 h because the hydrophilic outer shell MPEG prevented albumin adsorption on mixed micelles. This is one indication that mixed micelles could prolong the circulation after intravenous injection.
Two functional end-capped diblock copolymers galactosamine (Gal)-PEG3400-PLA830 (Gal-PEG-PLA, [Gal]: [PEG]: [LA]=8.4.:7.6:84 mol/mol) and fluorescein isothiocyanate (FITC)—PEG3400-PLA830 (FITC-PEG-PLA, [FITC]: [PEG]: [LA]=4:8:88 mol/mol) were synthesized by thiol-maleimide coupling reaction.
FITC-PEG-PLA Diblock Copolymer Synthesis. PLA-NH2. N-Boc-L-alaninol was converted to the corresponding alkoxide (N-Boc-L-alaninol-OK) using potassium/naphthalene. D,L-lactide (2 g) was then polymerized at 100° C. for 12 h using N-Boc-L-alaninol-OK (0.35 g) as an initiator and toluene (2 mL) as the solvent to obtain PLA-NHBoc. The polymerization was terminated by adding acetic acid to the reaction mixture and PLA-NHBoc was precipitated from diethyl ether. The Boc group was removed from the PLA-NHBoc (2.1 g) by treating with a mixed solvent of formic acid (20 mL) and CHCl3 (20 mL). After 9 h treatment at room temperature, the solution was poured into a large amount of diethyl ether to obtain the precipitate. The precipitate was vacuum dried at room temperature. The product (1.5 g) was then deprotonated in a mixed solvent of triethylamine (20 mL) and CHCl3 (20 mL) at room temperature for 8 h. PLA-NH2 was purified by a method similar to that for PLA-NHBoc. PLA-SH. Thiolated PLA was synthesized by covalent modification of the primary amino groups of PLA-NH2 by adding sulhydryl moieties. For the synthesis, PLA-NH2 (2 g) was dissolved in acetonitrile (10 mL) and then reacted with an excess of 2-iminothiolane hydrochloride (0.458 g) at room temperature for 15 h. The unreacted 2-iminothiolane was removed by repeated dialysis against 5 mM HCl solution followed by 1 mM HCl solution for 24 h each. The purified PLA-SH was vacuum dried. Maleimide-PEG-NH2. Aliquots of N-Methoxycarbonylmaleimide (0.2 g) in dimethyl sulfoxide (DMSO) (10 mL) were added to an aqueous solution of polyoxyethylene bis(amine) (1 g) at room temperature. The mixture was allowed to react for 6 h. After the reaction, the resulting Maleimide-PEG-NH2 was purified by recrystallization from a mixed solvent of dichloromethane and diethyl ether (1/1 vol/vol) at −20° C. PLA-PEG-NH2. PLA-SH (0.8 g) was dissolved in 0.1 M Tris/acetonitrile (1/3 v/v) (aq, pH 6.5, adjusted by 0.5 M NaOH solution) (15 mL) and then added to Maleimide-PEG-NH2 (2 g) Tris solution (10 mL). The reaction mixture was shaken and allowed to continue for 6 h at room temperature. After the reaction, the product was purified by dialysis against PBS and Milli-Q water using a cellulose membrane bag (molecular weight cut-off, 6000-8000; obtained from SpectrumLabs, Inc.) and then frozen in a freeze dryer system (Heto-Holten A/S, Denmark) to yield dried product. The dried product was dissolved in dichloromethane and purified by precipitation from diethyl ether to remove unreacted PLA-SH. NH2—PEG-PLA was obtained under vacuum. FITC-PEG-PLA. NH2—PEG-PLA (1 g) was dissolved in methanol (40 mL) and the fluorescein isothiocyanate (FITC) (0.15 g) was then added. The mixture was stirred for 24 h at room temperature. The reaction mixture was then dialyzed against 0.5 M NaCl solution followed by dialysis against Milli-Q water for 2 days to remove methanol solvent and unreacted small molecules. Dried FITC-PEG-PLA product was obtained by a freeze dryer system.
Gal-PEG-PLA Diblock Copolymer Synthesis. Gal-Maleimide. Aliquots of N-Methoxycarbonylmaleimide (0.68 g) in dimethyl sulfoxide (DMSO) (10 mL) were added to an aqueous solution of galactosamine hydrochloride (0.5 g) at room temperature. The mixture was allowed to react for 6 h. The resulting Gal-Maleimide was purified by precipitation from diethyl ether. PLA-PEG-SH. Thiolated PLA was synthesized from the PLA-PEG-NH2 with the addition of sulhydryl moieties. For the synthesis, PLA-PEG-NH2 (1 g) was dissolved in acetonitrile (15 mL) and reacted with an excess of 2-iminothiolane hydrochloride (0.1 g) at room temperature for 15 h. Unreacted 2-iminothiolane was removed by repeated dialysis against 5 mM HCl solution followed by 1 mM HCl solution for 24 h. The Milli-Q water was replaced every 3 h. The purified PLA-PEG-SH was vacuum dried. Gal-PEG-PLA. PLA-PEG-SH (1 g) was dissolved in methanol (15 mL) and Gal-Maleimide (0.1 g) was then added. The mixture was stirred for 24 h at room temperature. The reaction mixture was then dialyzed against 0.5 M NaCl solution followed by dialysis against Milli-Q water for 2 days to remove methanol solvent and unreacted small molecules. The dried Gal-PEG-PLA product was obtained by a freeze dryer system.
Multifunctional micelle incorporated with Dox was prepared using the dialysis method. First, Dox was neutralized with a 1.2 molar excess of triethyl amine in DMSO/DMF (4/1 v/v). This mixture was stirred to dissolve the drug. Fifty mol % of Graft I, 20 mol % of Block IV, 15 mol % of Gal-PEG-PLA, and 15 mol % of FITC-PEG-PLA were then dissolved in the drug solution. The mixture was dialyzed against Milli-Q water for 72 h using a membrane with a molecular-weight cut-off of 6000-8000 at room temperature. The Milli-Q water was replaced every 3 h. Multifunctional micelles were obtained by a freeze-drying process. The DOX loading level was about 31 wt % in weight, which was determined by a UV/Vis spectrophotometer as multifunctional micelles dissolved in DMSO.
To evaluate the effects of stimulus-response behavior on controlled drug delivery, the in vitro drug release behaviors of the Dox-loaded multifunctional micelles were studied in two different buffered solutions (pH 7.4 and 5.0).
Multifunctional micelles without Dox were also prepared for four components, including FITC-PEG-PLA, Gal-PEG-PLA, Block IV (mPEG5000-PLA1750) and Graft I copolymers by repeating the procedures of the preparation of the Dox-loaded multifunctional micelles except that Dox was not used. The graft copolymer (Graft I) in the multifunctional micelles could encapsulate anticancer drugs, and control drug release in response to pH or temperature changes. Block IV in micelles helped control the core-shell structure and obtain uniform micellar distribution. The fluorescence dye conjugated diblock copolymer FITC-PEG-PLA in micelles provided direct evidence of where micelles accumulated after cell uptake. On the other hand, the targeting moiety (Gal) conjugated diblock copolymer Gal-PEG-PLA could combine with the asialoglycoprotein of HepG2 cells in the active tumor targeting.
The Dox-loaded multifunctional micelles and free Dox were tested for in vitro cytotoxicity using a tetrazolium dye (MTT) method. The MTT-based cytotoxic activities of the Dox-loaded multifunctional micelles and free DOX were compared after 24 h and 72 h incubation with HeLa cells. The inhibition concentration (IC50) of the Dox-loaded multifunctional micelles was 25 μg/1 mL at 24 h but decreased to 4 μg/mL at 72 h. The cytotoxicity of the Dox-loaded multifunctional micelles at 72 h was similar to free Dox (IC50=1.2 μg/mL). On the other hand, the IC50 Of empty multifunctional micelles was 792 μg/mL after 72 h of incubation. This indicates that the cytotoxicity of HeLa cells came from the Dox released by the Dox-loaded multifunctional micelles.
To evaluate the functionality of the Dox-loaded multifunctional micelles in biomarker applications, confocal laser scanning microscopy (CLSM) was used to observe the fluorescence images of the Dox-loaded multifunctional micelles and released Dox after HeLa cells uptake (for 6 h incubation). The triggering mechanism of most particulate carriers must occur in the endosome to release the drug in the cytoplasm. The CLSM fluorescence images show that HeLa cells showed green fluorescence in the cytoplasm, indicating that the multifunctional micelles were located there. Additionally, the released Dox, with a red fluorescence, was localized in both the cytoplasm and the nucleus. The clear pathway of where particulate carrier delivery was observed by the FITC-labeled micelles.
To evaluate the functionality of multifunctional micelles in specific tumor targeting, the Dox-loaded multifunctional micelles prepared in Example 5 and Dox-loaded mixed micelles prepared in Example 2 were incubated with HepG2 (hepatocellular carcinoma) cells.
Tumor Targeting Evaluation. HepG2 cells (2×104 cells/mL) were seeded in a 25-T flask of DMEM medium with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37° C. After the HepG2 cells had been incubated in a logarithmic growth phase, the Dox-loaded multifunctional micelles or the Dox-loaded mixed micelles were added for 2 h at 4° C. HepG2 cells were twice washed by PBS solution, and fresh medium was added for 24 h and 48 h incubation in a humidified atmosphere of 5% CO2 at 37° C. At the end of the experiment, cell viability was calculated by trypan blue staining using a phase contrast microscopy (the positive control). The same process was repeated at 37° C. through the entire process as a negative control. The procedures were repeated except that galactose (150 mM) was also added to the system for performing an inhibition assay.
Hepatocytes have large numbers of asialoglycoprotein receptors on their surface that recognize galactose residues. Because of their specific ligand-receptor binding, the internalization of the Dox-loaded multifunctional micelles (containing the targeting moiety, Gal) into cancer cells can be performed by the receptor-mediated endocytosis process (active tumor targeting) and delivered to the lysosomes. The viability (percentage of surviving cells) of HepG2 cells after 24 h and 48 h incubation of the Dox-loaded multifunctional micelles was compared with the Dox-loaded mixed micelles.
It can be seen from Examples 5 and 6, multifunctional micelles encapsulating Dox were successfully prepared by dialysis, which can be used as cancer diagnosis agents and cancer drug delivery carriers. TEM images reveal that the Dox-loaded multifunctional micelles are spherical in shape and about 160 nm in size, which is suitable for intravenous injection and close to the typically required size under physiological conditions. Tumor targeting assay and CLSM measurements reveal that the Dox-loaded multifunctional micelles exhibit a high cytotoxicity by receptor-mediated endocytosis and show clear fluorescence imaging of their distribution. This shows a proof-of-concept: that is, producing an ideal micelle with a long circulation time, tumor recognition, and combined cancer diagnosis and controlled drug delivery for cancer therapy. It is apparent that multifunctional micelles combined with a near IR dye (e.g. Cy5.5) to replace FITC can be extended to animal models to evaluate the distribution in the body and cancer therapy.
Number | Date | Country | |
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60848382 | Oct 2006 | US | |
60848381 | Oct 2006 | US |