Ben Wang
benwang83@gmail.com
Gald to Join Jag Team!
Research Interests:
Single cell manipulation based on a multi-disciplinary approach for biological engineering, such as tissue engineering and cancer therapy.
Experience & Skills:
Chemistry, biology and engineering.
Project Pages: (Help)
DATE CREATED: 02/24/2009
ACTION PLAN
Meeting Summary (Help)
Meeting Date: 01/26/2009
Summary: Single cell coated with nanofibers by electrospining
Action Plan:
Progress on issues from last meeting:
Meeting Date: 01/26/2009
Summary: Macrophage encapulated with MMP-sensitive hydrogel for cancer therapy
Action Plan:
Progress on issues from last meeting:
PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)
PAPER TITLE
High-selective tumour-targeting drug delivery system based on matrix metalloprotease nanoparticles
A) Background
B) Hypothesis
B) Specific Aims
C) General Experimental Approach (Design etc)
D) Pitfalls and alternatives
E) Potential Figures
F) Future Directions
A) BACKGROUND:
B) HYPOTHESIS
C) SPECIFIC AIMS
AIM 1 -
AIM 2 -
Cancer is the most challenging and intractalbe issue in the medicine of our era. Chemotherapy as the primary cancer treatment has played significant roles in the past time. However, side effects is fatal problem because of the virulence of the anti-tumor drugs for normal tissues and cells. Novel delivery systems involved the identification of precise targets and site-specific drug release could enable new and better chemotherapeutic regimens making full advantage of existing pharmaceuticals. Matrix metalloproteinases (MMPs) are proteolytic enzymes and their basic mechanism of action — degradation of proteins — regulates various cell behaviours with relevance for cancer biology. MMPs can regulate the tumour microenvironment, and their expression and activation is increased in almost all human cancers compared with normal tissue. Based on property of MMPs, here we show a high seletive drug delivery system for breast cancer therapy. Using Particle Replication In Non-wetting Templates (PRINT), we fabricated monodisperse shape-specific 200 nm particles of MMP-2 and MMP-9 sensitive hydrogel with Dextran-peptide-methotrexate conjugates. The enzymatically-triggered smart nanoparticles should be proved to be a potential means for tumor-targeting and minimizing the side effects.
D) GENERAL EXPERIMENTAL APPROACH
E) PITFALLS AND ALTERNATIVES
F) ANTICIPATED FIGURES FOR PAPER or when you have data, FIGURES FOR PAPER
G) FUTURE DIRECTIONS
H) MATERIALS AND METHODS
Synthesis of MMPs-sensitive hydrogel
PEG-OH. Nominally four-arm PEGs and other multiarm PEGs were purchased from Shearwater Polymers (Huntsville, AL). These molecules consisted of a poly(glycerol) backbone with multiple PEG arms attached to it through an ether bond: 4arm-PEG-OH, molecular weight 10 kDa (nominal) (Mn ) 9270, Mw ) 9450); 4arm-PEG-OH, molecular weight 15 kDa (nominal) (Mn ) 12 120, Mw ) 15 600); 4arm-PEGOH, molecular weight 20 kDa (nominal) (Mn ) 19 400, Mw ) 20 210). In addition to the 4arm-PEG-OH macromers, 3arm-PEG-OH, mololecular weight 15 kDa (nominal), and 8arm-PEG-OH, molecular weight 40 kDa (nominal), were used.
Synthesis of PEG Vinyl Sulfone (PEG-VS). Multiarm PEG-VSs were synthesized by coupling PEG-OHs with an excess of divinyl sulfone (Aldrich, Buchs, Switzerland), in contrast to a previously published synthesis of PEG-VS via PEG-chloroethyl sulfone. PEG-OH (ca. 5 g) was either used as received and dissolved directly in 300 mL of dry dichloromethane (previously dried over molecular sieves) or, in some cases, PEG was dried by azeotropic distillation in toluene using a Dean Stark trap before starting the reaction. To the PEG dissolved in dichloromethane, NaH was added under argon, at 5-fold molar excess over OH groups. After hydrogen evolution, divinyl sulfone was added very quickly at 50- to 100-fold molar excess over OH groups. The reaction was carried out at room temperature for 3 days under argon atmosphere with constant stirring. Afterward the reaction solution was neutralized with concentrated acetic acid, filtered through paper until clear, and reduced to a small volume (ca. 10 mL) by rotary evaporation. PEG was precipitated by adding the remaining solution dropwise into ice-cold diethyl ether. The polymer was recovered by filtration, washed with diethyl ether, and dried under vacuum. The dry polymer was then dissolved in 200 mL of deionized water containing ca. 5 g of sodium chloride and extracted three times with 200 mL of dichloromethane. This solution was dried with sodium carbonate, and the volume was again reduced by rotary evaporation. Finally, the product was reprecipitated and thoroughly washed with diethyl ether to remove all remaining divinyl sulfone. The final product was dried under vacuum and stored under argon at -20°C. Derivatization was confirmed with 1H NMR (CDCl3): 3.6 ppm (PEG backbone), 6.1 ppm (d, 1H, =CH2), 6.4 ppm (d,
1H, =CH2), and 6.8 ppm (dd, 1H, -SO2CH=). The degree of end group conversion, as shown by NMR, was found to range from 95 to 98%. Gel permeation chromatography was used to confirm that the starting material (PEG-OH) and the end-functionalized PEG-VS have identical molecular weight distributions.
Peptide Synthesis, Purification, and Measurement of Reduced Thiol Content. Peptides were synthesized on solid resin (Novasyn TGR, Novabiochem, Laeufelfingen, Switzerland) using a Perspective Biosystems (Farmington, MA) Pioneer peptide synthesizer with Fmoc/HBTU/HOBT chemistry. All amino acids and activators were from Novabiochem; solvents were from Applied Biosystems (Rotkreuz, Switzerland). Peptides were cleaved from the resin for 6-8 h at room temperature using 8.8 mL of trifluoroacetic acid (Fluka), 0.5 mL of deionized water, 0.5 mL of phenol (Aldrich), and 0.4 mL of triisopropylsilane (Aldrich) per gram of resin. The resin was removed by filtration, and the solution was precipitated in cold diethyl ether, recovered by filtration, and dried under vacuum. Peptides were purified by C18 chromatography (Prep Nova-Pak HR C18 6 μm, 60 Å, 19 _ 300 mm column, Waters, Milford, MA) using a Perseptive Biosystems Biocad 700E. The purified peptide was analyzed by using a Voyager Elite (Perseptive Biosystems) matrix-assisted laseer desorption ionization time-of-flight mass spectrometry. Peptides were stored under argon at -20°C.
Two peptides were synthesized with the N-terminal amino acid being a N-acetylglycine: Ac-GCRD-GPQG↓IWGQDRCG (molecular weight 1745.9 g/mol) and Ac-GRCRGPQG↓IWGQ- RCRG (mol. weight 1828.1 g/mol). The sequence in the middle (in italics) was chosen on the basis of sensitivity to matrix metalloproteinases (MMPs). This protease family is extensively involved in tissue development and remodeling. Here, MMP substrates were structurally derived from a sequence found in the R(1)-chain of human (also chick and calf) type I collagen, namely, the sequence GPQG↓IAGQ (↓ indicates the cleavage site). Simple mutations in this sequence lead to wide variations in degradation kinetics; the sequence GPQG↓IWGQ, one of the fastest degrading substrates for several MMP members, was employed in the present work as a representative example. The sulfhydryl content of both peptides was measured using Ellman’s reagent (5,5'-dithio-bis(2-nitrobenzoic acid), Sigma). Forty milligrams of Ellman’s reagent was dissolved in 10 mL of 0.1 M phosphate buffer, pH 8.0. One hundred micrograms of this solution was added to 3 mL of 0.1 M phosphate buffer, pH 8.0, containing 0.1 μmol of thiol-containing peptide. The thiol concentration was estimated using an extinction coefficient of 14150 M-1 m-1 at 412 nm. The amount of free thiols as measured for peptides from several different syntheses was between 75 and 95% (accuracy ca. (5%) and changed significantly from batch to batch. Sometimes relatively low values were obtained that may be attributed to the presence of remaining salt from the peptide synthesis (i.e., a weighing error due to counterions from the cleavage of the peptide from the resin) or a certain fraction of the -SH groups being in a nonreactive oxidized form (-S-S-). For this reason, experiments were performed with one peptide batch with 90% free sulfhydryl groups.
Hydrogel Formation. Hydrogels were formed by Michael-type addition of thiol-containing peptides onto multiarm PEG-VS. Anti-tumor drug (Methotrexate) was dispersed in solution. Each precursor was dissolved in 0.3 M triethanolamine solution at a desired pH and solid concentration (w/v). For example, to make stoichiometrically balanced 10% (w/v) gels, 10 mg (e.g.) of 4arm-PEG-VS, 20 kDa, was dissolved in 89.9 μL and mixed with a precursor solution containing 1.7 mg of the peptide Ac-GCRD-GPQG↓IWGQDRCG in 18.2 μL of the same buffer, giving the final volume of ca. 117.5 μL of reaction solution (i.e., the increase in volume due to the presence of the dissolved reaction precursors was taken into account). Noteworthy, problems in reproducibility of network properties may arise primarily from weighing errors. For this reason, only relatively large amounts of peptides (ideally >2 mg, to reduce the maximum weighing error to 5%) were weighed and used to make a large series of gels. Alternatively, to achieve the best results, large quantities of peptide should be dissolved in deionized water, aliquoted, freeze-dried, and stored at -20°C under argon as a defined quantity of powder for further use. Immediately after mixing of the precursors, the solution was quickly vortexed and then transferred to the center of a hydrophobic glass microscope slide coated with SigmaCote (Sigma, treated according to the supplier’s instructions). Spacers (0.7 mm thick) were placed at the ends of the glass slide, and a second hydrophobic slide was placed on top. The two slides were clamped together with binder clips. The drop of reaction solution contacted only the hydrophobic glass and spread to form a circular disk with a thickness of 0.7 mm. Gelation occurred within a few minutes at 37°C in a humidified incubator; however, the cross-linking reaction was carried out for additional 1-2 h to achieve optimal crosslinking efficiency.
Preparation and characterization of PRINT nanoparticles
Preparation. The fabrication of patterned Fluorocur™ molds has been described briefly, 20 mL of Fluorocur™ resin containing 0.1% (w/w) of 2,2-diethoxyacetophenone was pooled in the center of an 8 inch patterned master (with feature sizes of 200 nm) which was set up inside an enclosed UV chamber. Ten minutes was allowed to pass so that the Fluorocur™ resin was spread out over the entire 8 inch wafer. The entire system was then purged with nitrogen for 3 minutes. Following this, the coated wafer was exposed to UV irradiation (λ = 365 nm, power > 20 mW/cm2) for 2 minutes to cure the Fluorocur™ resin. The elastomeric mold was then removed from the master template by gently peeling it away from the silicon surface. In these experiments, the PRINT particles were derived from a mixture composed of 78% (w/w) PEG triacrylate, 20% (w/w) PEG monomethyl ether monomethacrylate, 1% (w/w) 2,2-diethoxyacetophenone, and 1% (w/w) para-hydroxystyrene. A 10% (w/v) solution of this mixture in 2-propanol (filtered through a 0.22 μm PTFE filter) was prepared. This solution (1 mL) was then sprayed onto a Fluorocur™ patterned mold using an air brush and residual 2-propanol was allowed to evaporate over 10 minutes. A poly(ethylene) sheet (American Plastics Co.) was then placed over the 8 inch (diameter) mold ensuring that the entire active area was covered. This poly(ethylene) sheet was then peeled back at a rate of approximately 2.5 cm/min. Following this, the mold was placed in a UV curing chamber. The chamber was purged with nitrogen for 3 minutes and UV irradiation was applied (λ = 365 nm, power > 20 mW/cm2) for 2 minutes.
To facilitate removal of the particles from the mold, a physical means for harvesting the particles was utilized. Specifically, a 2 mL aliquot of acetone (filtered through a 0.22 μm PTFE filter) was placed on the particle-filled mold and this drop of acetone was gently moved along the surface of the mold using a glass slide. The movement of the glass slide facilitated release of the particles from the mold. The suspended particles were collected in a 50 mL Falcon tube and diluted to the 50 mL mark with filtered acetone after particle collection was complete. The suspension was vortexed for 10 minutes and was centrifuged at 3200 rpm for 30 minutes using a IEC CENTRA CL2 Centrifuge (Thermo Electron Corporation). The supernatant was removed via aspiration and the particle pellet was redispersed in 50 mL of fresh acetone by vortexing for 10 minutes followed by centrifugation for an additional 30 minutes. This process was repeated once more and after aspiration the particles were redispersed in 5 mL of distilled water by sonicating the dispersion for 15 minutes. The particle dispersion was filtered through a 20 μm filter into a fresh 50 mL Falcon tube, and diluted to the 50 mL mark with acetone. This particle suspension was then centrifuged for one hour. The supernatant was removed via aspiration and the particle pellet was redispersed in 50 mL of fresh acetone by vortexing for 10 minutes followed by centrifugation for an additional 30 minutes. This washing process was repeated once more (with acetone) and after aspiration the particles were redispersed in a minimal amount of acetone, transferred to a tarred Eppendorf tube, and centrifuged in a microfuge (Fisher Scientific) for 20 minutes. The supernatant was removed and the pellet was dried in a vacuum oven overnight, massed, and dispersed in the appropriate amount of sterile water to make a 10 mg/mL dispersion of particles.
Characterization. Analysis using scanning electron microscopy and dynamic light scattering. Zeta potential measurements.
In vitro cytotoxicity
Drug release effect of MMPs (2 & 9) treatment on nanoparticles
Time and concentration.
Investigation of targeting effect of nanoparticles
Co-cuture cancer cells & normal cells with nanoparticles (with drug) and cell viability.
I) RESULTS
Fig. 1 Illustration of PRINT process. a, Fabrication of the silicon master template; b, Wetting of the silicon master with liquid fluoropolymer, followed by curing; c, PFPE elastomeric mold produced with nanoscale features from the master; d, Confining organic liquid to cavities by applying pressure between mold and aPFPE surface; e, Removal of organic particles from mold with adhesie layer; f, Dissolution of adhesive layer producing free particles.
Fig. 2 Results of the PRINT process. a, SEM image of the original trapezoidal silicon master (200 nm feature size) used to generate the PFPE mold that was used to generated the 200 nm particles. b, SEM image of 200 nm MMP-sensitive hydrogel particles. c, Representative AFM image of replicate PEG particles. d, Fluorescent confocal micrograph of 200 nm nanoparticles containing methotrexate with * fluorescent dye. e, A diameter study of the particles harvested using dynamic light scattering.
Fig. 3 Cytotoxicity of nanoparticles and the products of degradation against the normal cells and breast tumor cells.
Fig. 4 Enzymatic degration curves of the MMP-2 and MMP-9 sensitive hydrogel nanoparticles and Physicochemical release kinetics of the anti-tumor drug (methotrexate).
Fig. 5 Effect of nanoparticles, methotrexate, nanoparticles with methotrexate on the normal cell and the breast cancer cell.
J) DISCUSSION
K) LITERATURE