Patent Application
20130131151
1. Technical Field
The present invention relates to a method for photopolymerizing hydrogel using X-ray irradiation, and more particularly, to a method for photopolymerizing hydrogel in vivo using X-ray irradiation.
2. Background
The fabrication of 3D biomaterial implants has been the subject of much research due to their potential for a broad range of biomedical applications. Such 3D implants can serve as temporary scaffolds and promote cell reorganization and the formation of biofunctional substitutes. Hydrogel has also attracted much attention particularly for use in local delivery of matrix-encapsulated proteins and nucleic acids to chondrocytes, fibroblasts, vascular smooth muscle cells, osteoblasts, neural precursor cells and stem cells for regenerative therapies, since many biophysical properties of Hydrogel are similar to those of soft biological tissues. Hydrogel can also be used to obtain flexible 3D formations, mechanical stability and a good tissue culture environment for biomedical applications.
Notwithstanding the above-mentioned positive points, a serious obstacle remains to be solved for the wide application of hydrogel: the need for accurate administration in vivo. Inserting a solid material, even gel-like, into a live system is difficult without undesirable surgery. While many different approaches exploiting a phase change after injection from a more easily handled liquid to a gel have been explored. Hydrogel is one of the most biocompatible materials exhibiting such a phase change. Of particular interest in this context, beyond passive implantation, is the local activation of cell-material interactions.
The practical implementation of this approach requires injectable precursors and an initiator that stimulates polymerization at precise locations deep in the tissue. Low-viscosity precursor hydrogel solutions can be quite easily injected by syringe with the appropriate mixture of cells and bioactive factors, and placed at the desired 3D location, to be then polymerized by the initiator. Accurate initiator administration is a much more serious problem. Typical polymerization initiators include heat, chemicals, mechanical factors, ultrasound and photons, all of which are difficult to accurately administer in vivo. Recently, it was found that glucose oxidation and Fe2+ generate hydrogel within minutes at room temperature and ambient-pressure oxygen. This was found to yield cellular encapsulation into hydrogel scaffolds, but the reaction rate must be improved for in vivo administration. Another approach, shear thinning, produced gelation in vivo, but also requires administration in vivo via a long circulation path, beyond local injection, for which mechanical factors cannot be easily controlled and monitored.
Photopolymerization by visible or ultraviolet (UV) light exploiting photoinitiators to produce free radicals, which subsequently initiate polymerization through active sites on macromeric chains, could solve the problem. The space and time characteristics of the polymerization process can be controlled by the shape and intensity of the light beam as well as by the illumination time. The polymerization rate can be high enough to produce hydrogel with a short exposure (seconds to tens of seconds).
Photopolymerization is already used for biomedical hydrogel production. Under standard protocols, the acrylate-terminated monomer undergoes photopolymerization by exposure to light in the presence of appropriate photoinitiators. It has been shown that UV and visible light polymerize hydrogel in vivo for cell encapsulation applications requiring high biocompatibility during the polymerization process without adversely affecting the encapsulated cells. Another example of successful in vivo application is transdermal photopolymerization, again using UV and visible light, which opens the door to minimally invasive hydrogel implantation. However, many photoinitiators, particularly those with UV or visible absorption, have some water solubility and cytotoxicity problems. Furthermore, light scattering and absorption limit the use of UV or visible photopolymerization where accurate local control of the implantation is required. These problems, linked to the administration depth and to shadowing, stimulated the development of light-independent encapsulation systems for cell-laden scaffolds.
Thus far, none of the above approaches has been fully satisfactory in accurately polymerizing the scaffold leading to the desired 3D shapes in vivo. Also note that a high polymerization rate in vivo is required for the hydrogel to maintain its shape, location and functions without complications due to the biological response.
One aspect of the present invention provides a method for photopolymerizing hydrogel in vivo using X-ray irradiation.
A method for preparing a hydrogel according to this aspect of the present invention comprises the steps of injecting a precursor with at least two alkene groups into a predetermined portion, injecting at least one specie into the predetermined portion, and performing an X-ray irradiation on the predetermined portion to induce a polymerization reaction of the precursor to form a porous hydrogel with the specie embedded inside the porous hydrogel. In one embodiment of the present invention, the specie is selected from the group consisting of nucleic acid and adhesion agent.
The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter, and form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures or processes for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.
The objectives and advantages of the present invention are illustrated with the following description and upon reference to the accompanying drawings in which:
The inventors tested an alternate method and demonstrated its capability to produce accurate 3D hydrogel implants without surgery. The key factor was polymerization of PEG diacrylate (DA) by short-wavelength, high penetration irradiation, wherein the process was substantially stimulated in situ by X-rays. These tests open the way to a potentially very flexible approach, since X-rays can be locally controlled so that the implant could in principle be “written” in 3D with excellent accuracy. Similar performances were demonstrated for X-ray lithography by combining multi-directional irradiation and variable masks; this enabled in particular the accurate fabrication of 3D structures such as photonic crystals. Since hydrogels are extensively used for the local delivery of proteins and other similar applications, the inventors also tested procedure to obtain hydrogel containing factors for gene delivery.
The inventors selected PEG DA-based hydrogel for several reasons. The PEG segment of PEG DA is non-immunogenic, non-toxic, and highly hydrophilic. The use of PEG for drugs was demonstrated to be safe and effective in many US Food and Drug Administration-approved therapeutic procedures. PEG is also extensively used as a coating substance to obtain biomaterial surfaces that resist non-specific protein adsorption and cell adhesion.
The approach schematically presented in
The inventors first performed a subcutaneous injection (SC) in mice of the precursor solution including PEG DA and PBS (phosphate buffered saline) at the optimal concentration ratio. The exposure to X-rays triggered the in situ polymerization and hydrogel formation. The remaining non-polymerized solution was then physiologically eliminated from the subcutaneous tissue. The testing was extended to the photopolymerization of PEG DA-based hydrogels with heparin or PEI/DNA and PEI/siRNA nanoparticles and to the corresponding properties. The tests also identified measures that can increase the polymerization rate and reduce the risk of radiation effects. Such measures are based on the addition of Au nanoparticles in the precursor solution and could be improved in the future from the use of microfocused X-rays and from the development of even more X-ray sensitive biopolymers. With such improvements, the radiation dose could be reduced sufficiently to allow cell encapsulation procedures.
To assess the ability of PEG DA to polymerize under X-ray irradiation (
Compared to other hydrogel polymerization approaches, such as UV-induced polymerization and chemical crosslinking, the conversion rate by X-rays is quite reasonable. Note that this rate is limited by the need to use a safe X-ray dose. The dose, however, can be reduced by increasing the conversion rate; this appears feasible using materials with greater X-ray sensitivity and/or micro-focused X-rays that can increase the local intensity at selected points by orders of magnitude.
To evaluate the stability of the polymerized hydrogels, their weight loss (%) is measured over time in PBS solution at 37° C. For the assay, the hydrogels are produced in porous transparent cylinder containers (10 mm diameter, 5 mm height) (
Compared to other polymerization approaches, X-ray synthesized hydrogel is quite stable in terms of long-term weight loss and swelling ratio. This can be attributed to the less complex and uniform chemistry involved in the polymerization process.
The testing was extended to the effects of the hydrogel on human HT-1080 fibroblast cells. The cell viability was determined by the MTS assay after 24 hours incubation with PEG DA at different concentrations, using untreated cells as the reference. PEG DA with 15.5, 18.6 and 23.3 mg mL−1 concentration resulted in an average cell viability of greater than 80% (
The toxicity tests are of course essential for the practical applications of the procedure. In our case, it is insufficient to merely consider the toxicity of the precursors (which are certified biomaterials) and of the hydrogel: one must also analyze their combined effects. Our testing verified that the X-rays did not alter the precursor or the hydrogel and produced no toxicity effect.
The adhesion of cells to the hydrogel is an important mechanical property that could affects its application. In our tests, we found that the HT-1080 cells on our PEG DA-based hydrogels had an abnormal spherical morphology (
We tested the local delivery of plasmid DNA or small interfering RNA (siRNA) in nanoparticles by using PEI, which is widely exploited as a nucleic acid carrier. We found that the use of PEG DA-based hydrogel with PEI/plasmid DNA nanoparticles (
To decrease the X-ray dosage, we incorporated 20 mM bare gold nanoparticles of 15 to 20 nm diameter in the PEG DA solution before performing the x-ray irradiation tests. As a high Z material, Au strongly absorbs hard X-rays and can be expected to enhance their effects. The nanoparticle shape optimization can further increase this effect. In our tests, the precursor with gold nanoparticles was completely polymerized within 30 seconds, 3 times faster than without nanoparticles (
PEG DA (Mn=700) and branched poly(ethylene imine) (PEI, Mw=25,000) were purchased from Aldrich (Milwaukee, Wis.). Heparin sodium (100 KU) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma Co. (St. Louis, Mo.). CellTiter 96® AQueous one solution cell proliferation assay systems for the MTS assay were purchased from Promega (Madison, Wis.). The plasmid DNA (pEGFP-N2, 4.7 kb, coding an enhanced green fluorescence protein reporter gene) was purchased from Clontech (Palo Alto, Calif.). pEGFP-N2 was amplified using DH5α and purified by Qiagen Plasmid Mega Kit (Germany) according to the manufacturer's instructions. The purity of plasmids was analyzed by gel electrophoresis (0.8% agarose), while their concentration was measured by UV absorption at 260 nm (Jasco, Tokyo, Japan). The siRNA duplex targeting enhanced green fluorescence protein (EGFP, GFP-22 siRNA) was purchased from Qiagen (Germany), and the siRNA sense sequence was EGFP, 5′-GCAAGCUGACCCUGAAGUUCAUdTdT-3′.
The experiments were performed on Beamline 01A at the National Synchrotron Radiation Research Center (BL01A NSRRC, Hsinchu, Taiwan). Six silicon wafers (thickness: 550 μm) were used to reduce the dose rate from 5.10 kGy s−1 to 110 Gy s−1 for the sample volume (10×10×10 mm3). The precursor solution was obtained by dissolving PEG DA in 0.6 mL deionized (D.I.) water; the volume ratio was 1/1, 1/3, 1/5, 1/10, 1/25 or 1/30. The solution contained no photoinitiator and the photopolymerization was achieved by exposure to the X-ray beam for 30, 60, 90, or 180 seconds.
The characterization included FTIR with a Perkin-Elmer Spectrum One FTIR instrument (the substrate being a silicon wafer) and 1H-nuclear magnetic resonance (NMR) spectroscopy with a Varian Unity Inova 500 MHz spectrometer. A 99.8% pure DMSO-d6 solution was used as the reference. The following peaks consistent with the proposed structure of PEG DA-based hydrogel were observed: δH (500 MHz; DMSO-d6, ppm): 2.4-2.6 (4H, —C(O)CH2CH2—), 3.7, (4H, —OCH2CH2—).
The surface morphology of our hydrogels was examined by scanning electron microscopy (SEM, JEOL, JSM-5600, Tokyo, Japan).
We analyzed the degradation of the synthesized hydrogels by first measuring their dry weight (Wi) after permanence for at least 24 hours after photopolymerization in a vacuum oven. The dried hydrogels were then incubated in 50 mL of PBS solution at 37° C.; the PBS solution was replaced daily. After a certain time, the hydrogels were removed, rinsed with PBS solution, vacuum-dried and weighed obtaining a dry weight value Wd. The relative weight loss (%) was calculated as (Wi−Wd) Wi−1.
To determine the equilibrium swelling ratio of the hydrogels, their dry weight was measured immediately after photopolymerization, then they were allowed to swell in PBS at 37° C. for 1, 2, 3, 4, 5, 6 or 7 weeks; the PBS solution was again replaced daily. After the swelling period, the samples were rinsed with PBS, and the swollen hydrogel weight (Ws) was measured. The swelling ratio (Q) was calculated as Q=Ws Wd−1.
The molecules were heparin or PEI. In the first case, 300 mg of heparin sodium salt was dissolved in 3 mL D.I. water while stirring. The negatively charged heparin solution was added to the PEG DA solution (with a volume ratio of 1/25) reaching a concentration of 0.01, 0.02, 0.05, 0.1, 0.2 or 0.5 mg mL−1 and X-ray irradiated for 90 seconds.
For the inclusion of PEI-based nanoparticles, 10 mg of branched PEI with an average Mw of 25,000 was added to 10 mL of D.I. water. The solution was then filtered with a 0.2 μm Millipore (Billerica, Mass.) instrument and stored at 4° C. A nucleic acid (plasmid DNA or siRNA: 1 μg) was then diluted in 100 μL D.I. water and vortexed. After about 1 minute, the PEI and nucleic acid solutions were mixed and vortexed for 30 minutes. The N/P ratios for PEI/DNA and PEI/siRNA nanoparticles (defined as PEI nitrogen/nucleic acid phosphate (N/P)) were 10/1 and 8/1, respectively. The PEI-based nanoparticle solution was then mixed with 1/25 volume ratio PEG DA solution and photopolymerized by 90 seconds of X-ray irradiation.
Electron micrographs were obtained with a high-resolution transmission electron microscope, HRTEM, JEOL JEM-2100F. The samples were prepared by depositing 10 μL of PEI/DNA or PEI/siRNA nanoparticle solution on a carbon-coated copper grid and air-drying.
Human HT-1080 fibroblasts (ATCC, Manassas, Va.) were grown in Dulbecco's modified Eagle's medium (DMEM, Biosource, Rockville, Md.) with 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin at 37° C. in a humidified 5% CO2 atmosphere. 105 HT-1080 cells were seeded in each one of the wells of a 24-well plate and fed with complete DMEM for 12 hours. The cells were then exposed to PEG DA and PEG DA-based hydrogels at different concentrations (15.5, 18.6, 23.3, 46.6, 93.3 and 155.3 mg mL−1). The exposures were performed with different PEG DA/D.I. water volume ratios (1/3, 1/5, 1/10, 1/20, 1/25 and 1/30), and different heparin concentrations (0.01, 0.02, 0.05, 0.100, and 0.500 mg mL−1) and performed for 24 hours. The CellTiter 96® AQueous one solution cell proliferation assay system with the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) was used to measure the mammalian cell survival and cell proliferation. The optical density (OD) value of formazan at 490 nm quantified the cell viability. The normalized cell viability was calculated by comparing the absorbance of untreated cells to that of cells exposed to PEG DA or to hydrogels.
Effects on Cells of Hydrogel-Mediated Nanoparticles for Plasmid DNA or siRNA Transfection
Cells of the same type as those above were seeded and incubated in DMEM with 100 U mL−1 penicillin, 10% FBS, and 100 mg mL−1 streptomycin for 12 hr before transfection. Subsequently, a porous polyester Transwell™ insert (Corning, N.Y.) with pore size of 1.0 μm was placed above the cell monolayer (in one well of a 6-well dish) to separate the hydrogels with PEI-based nanoparticles (Transwell™ insert) and the target HT-1080 cells (6-well dish). The 3.5 mL of complete DMEM medium was added to the culture without removing the Transwell™ insert; the complete DMEM medium solution was replaced daily. For the siRNA tests, we used recombinant HT1080 cells with the constitutive EGFP and luciferase expression as described in the literature. The transfected cells were then directly observed by a confocal microscope (Olympus IX 70, Olympus). The cells transfected with PEI-based nanoparticles were stained overnight by propidium iodide (PI, Molecular Probes, Eugene, Oreg.) to label the nuclei.
The PEI/plasmid DNA or PEI/siRNA nanoparticles were incorporated in the PEG DA-based hydrogel, and their release was evaluated by measuring EGFP intensities over one week. Flow cytometry analysis of EGFP-transfected cells was conducted with a benchtop system (FACSCalibur, Becton Dickinson) equipped with a 488 nm argon laser and a band-pass filter at 505-530 nm to detect EGFP. Untransfected cells were used as the control. The cells were appropriately gated by forward and side scatters, and 10,000 events per sample were collected. The assays of gene expression and gene silencing were quantitatively analyzed over one week.
All procedures involving animals were approved by Academia Sinica Institutional Animal Care and Utilization Committee (AS IACUC). BALB/cByJNarl mice (20-25 g) were provided by National Laboratory Animal Center (Taiwan). All mice were housed in individually ventilated cages (five per cage) with wood chip bedding and kept at 24±2° C. with a humidity of 40%-70% and a 12-hour light/dark cycle.
We injected 50 μL of PEG DA solution (with 1/25 volume ratio with respect to PBS) into the subcutaneous mouse tissue. The mice were placed on a movable stage for X-ray irradiation lasting 800 milliseconds. During X-ray irradiation the mice were kept under anesthesia using 1% isoflurene in oxygen. In vivo biocompatibility was examined on day seven to evaluate the immune responses of the epithelial cells overlying the either PEG DA or in situ photopolymerization of PEG DA-based hydrogel in subcutaneous mouse tissue. After sectioning, tissue slice sections of 10-15 μm thickness from each animal were diagnosed on the basis of haematoxylin and eosin (H&E, Sigma-Aldrich, Mo., USA) staining, and imaged with a Nikon ECLIPSE TS100 microscope.
After synthesizing our hydrogel in vivo for 7 days, the mice (weight approximately 20-25 g) were sacrificed by intramuscular injection of Zoletil 50 (50 mg kg−1; Virbac Laboratories, Carros, France). Subcutaneous tissue portions removed were immersed in the 3.7% paraformaldehyde for 24 hours. After fixation, the tissue portions were washed by PBS solution three times for 1 hour. All tissues were dehydrated by subsequent immersions in ethanol solutions, from low to high concentration, and then embedded in paraffin. Tissue specimens were sliced to 10 μm thickness and immersed in Xylene three times for 5 minutes to remove the remaining wax. Afterwards, the specimens were H&E stained for optical microscopy imaging.
The bare Au nanoparticle solution of 20 mM was added to the PEG DA solution (with volume ratio of 1/25) reaching a concentration of 0.67 mM and X-ray irradiated for 30 seconds. The Au nanoparticle solution was prepared and characterized following a previously developed method of one-pot synthesis by intense X-ray irradiation.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. For example, many of the processes discussed above can be implemented in different methodologies and replaced by other processes, or a combination thereof.
Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
| Number | Date | Country | |
|---|---|---|---|
| 61560936 | Nov 2011 | US |
