The present invention generally relates to introducing genetic material to living cells. In some embodiments, the present invention relates to compositions of matter for targeted delivery of nucleic acids to cells. In other embodiments, the present invention relates to methods of targeted delivery of nucleic acids to cells. In still other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone. In yet other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone thereby resulting in biodegradability, and in some embodiments, controlled biodegradability.
Previously, non-viral vectors have been unable to reach the transfection efficiencies of viruses. Several non-viral vectors have incorporated inactivated virus particles, or fusogenic viral peptides, that lead to improved transfection efficiencies. Unfortunately, immunogenicity is still problematic. Therefore it is also desirable to have non-viral vector that is capable of carrying effective amounts of genetic material, and efficiently transfecting cells while avoiding deleterious side affects such as immune response.
United States Published Patent Application No. 2005/0025820 (hereinafter the '820 application) is directed to a method and system for systemic delivery of growth arresting, lipid-derived bioactive compounds. More specifically, the '820 application discloses delivering “gene therapy agents” using a variety of means including microspheres, and nanoparticles. The delivery means set forth in the '820 application include PEGylated liposomes, and inorganic nanoparticle shells. Furthermore, the '820 application discloses that the delivery means can be “targeted” to particular kinds of cells by coupling it with targeting moieties. In contrast, the present invention is directed to micro- and/or nano-capsules comprising poly(lactide-co-glycolide), L-tyrosine phosphate, or any combination thereof.
United States Published Patent Application No. 2002/0131995 (hereinafter the '995 application) is directed to targeted drug delivery with CD44 receptor ligand. More particularly, the '995 application discloses using a variety of delivery vehicles, including liposomes and microspheres, for delivering drugs to targeted cells and/or tissues. The '995 application also discloses using such vehicles for delivering a variety of drugs including DNA. Additionally, the '995 application discloses using hyaluronan or other glucosaminoglycans having an affinity for the CD44 receptor as targeting agents. Although the '995 application mentions that the delivery vehicle can be a microsphere, it does not disclose how to make such a microsphere, nor does it suggest what materials such a microsphere would comprise. Furthermore, the '995 application states that liposome embodiments are preferred, and it goes into substantially greater detail regarding how to make and use delivery vehicles made from liposomes.
In contrast, the present invention teaches micro- and/or nano-capsules comprising poly(lactide-co-glycolide), and/or L-tyrosine phosphate. Furthermore, the present invention also includes targeting agents other than hyaluronan as well as optional additives related to biocompatibility and nucleic acid transport, each of which further distinguishes the present invention from the '995 application.
United States Published Patent Application No. 2005/0037075 (hereinafter the '075 application) is directed to targeted delivery of controlled release polymer systems. More particularly, the '075 application discloses polymer systems such as micro and/or nano-spheres made from a variety of polymers. For example, Paragraph [0033] states:
Additionally, the '075 application discusses using nucleic acid ligands to target particular kinds of cells. Furthermore, the '075 application discusses using such targeted micro/nano-spheres for delivering nucleic acids to cells. The present invention includes micro- and/or nano-capsules comprising poly(lactide-co-glycolide) and/or L-tyrosine phosphate, at least one targeting agent, and optionally including PEG-g-chitosan, and/or polyethylenimine. Although the '075 application discloses using poly(lactide-co-glycolide) as micro and/or nanosphere delivery vehicles, it does not disclose poly(lactide-co-glycolide) in combination with PEG-g-chitosan and/or polyethylenimine. Furthermore, the '075 application is limited to nucleic acid targeting agents, whereas the present invention includes non-nucleic acid targeting agents. Furthermore, the present invention includes micro- and/or nano-capsules comprising L-tyrosine phosphate, which is not disclosed by the '075 application.
Thus, there is a need in the art for a non-viral vector that is capable of targeted delivery to selected cells and/or cell types in an organism, efficiently transfecting such cells, and avoiding deleterious side effects. Advantageously, a non-viral vector can mimic a virus's ability to enter and transfect a cell, and do so without eliciting an immune response or causing the replication of competent viruses. Also advantageously, a non-viral vector can be biodegradable, nontoxic, protect genetic material disposed therein from enzyme degradation, and/or be able to avoid endosome encapsulation. Furthermore, it is desirable to have a vector that does not trigger an immune, coagulation, and/or inflammatory response.
The present invention generally relates to introducing genetic material to living cells. In some embodiments, the present invention relates to compositions of matter for targeted delivery of nucleic acids to cells. In other embodiments, the present invention relates to methods of targeted delivery of nucleic acids to cells. In still other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone. In yet other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone thereby resulting in biodegradability, and in some embodiments, controlled biodegradability.
In one embodiment, the present invention relates to a composition for targeted delivery of nucleic acids to cells comprising: at least one synthetic polymeric micro- or nano-capsule, wherein the capsule comprises one or more materials selected from poly(lactide-co-glycolide), L-tyrosine polyphosphate, L-tyrosine polyurethane, or any combination thereof; at least one targeting moiety disposed on the surface of the capsule and available for binding to target molecules, wherein the targeting moiety comprises any moiety capable of specifically binding with target molecules such as antibodies, antibody fragments, antigens, transmembrane proteins, glycoproteins, and any combination thereof; an additive for enhancing biocompatibility selected from one or more of PEG-g-chitosan and amphiphilic PEG species; and an additive for assisting in DNA transport across a cell membrane selected from one or more of linear polyethylenimine, PEG-g-chitosan, and any combination thereof.
In another embodiment, the present invention relates to an L-tyrosine-based containing polymer compound selected from L-tyrosine-based polyphosphate polymers, L-tyrosine-based polyurethane polymers, or blends of two or more thereof wherein at least one L-tyrosine-based amino acid moiety, or derivative thereof, is present in the backbone of the polymer compositions.
In still another embodiment, the present invention relates to an L-tyrosine-based polyphosphate polymer compound comprising at least one polymer composition having a formula shown below:
wherein the number of repeating units, x, is selected so that the molecular weigh of the above L-tyrosine polyphosphate before degradation is approximately in the range of about 5,000 Da to about 40,000 Da.
In still yet another embodiment, the present invention relates to an L-tyrosine-based polyurethane polymer compound comprising at least one polymer composition having a formula shown below:
wherein the number of repeating units, m, n and p, are selected so that the molecular weight of the above polyurethane compounds are in the range of about 4,000 Da to about 1,000,000 Da.
In still yet another embodiment, the present invention relates to a method for producing at least one polyurethane polymer compound comprising the steps of: (i) providing at least one macrodiol, at least one diisocyanate and at least one chain extender; (ii) reacting the at least one macrodiol, at least one diisocyanate and at least one chain extender to form at least one polyurethane polymer compound; and (iii) collecting the at least one polyurethane compound, wherein the at least one chain extender contains L-tyrosine, or a functional derivative or functional moiety thereof.
In still yet another embodiment, the present invention relates to an L-tyrosine-based polyphosphate polymer compound comprising at least one polymer composition having a formula shown below:
wherein x is an integer in the range of about 10 to about 80.
In still yet another embodiment, the present invention relates to an L-tyrosine-based polyurethane polymer compound comprising at least one polymer composition having a formula shown below:
wherein n is an integer in the range of about 5 to about 25, m is an integer in the range of 1 to about 4, and p is an integer in the range of about 20 to about 200.
The present invention generally relates to introducing genetic material to living cells. In some embodiments, the present invention relates to compositions of matter for targeted delivery of nucleic acids to cells. In other embodiments, the present invention relates to methods of targeted delivery of nucleic acids to cells. In still other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone. In yet other embodiments, the present invention relates to polymers that contain at least one amino acid in their backbone thereby resulting in biodegradability, and in some embodiments, controlled biodegradability.
The present invention generally relates to methods and materials for targeted delivery of genetic material to eukaryotic cells. Some embodiments of the present invention include nanospheres formulated from a polymer blend of L-tyrosine polyphosphate (LTP), polyethylene glycol grafted to chitosan (PEG-g-CHN), and plasmid DNA (pDNA) complexed with linear 2 polyethylenimine (LPEI) as non-viral gene delivery vector. Thus, some embodiments are capable of mimicking a virus by being taken up by, for example, mammalian cells, escaping endosome entrapment, protecting genetic material disposed therein from enzyme degradation, and/or efficiently transfecting cell's in vitro.
Sustained Release from Non-Viral Vectors:
Non-viral vectors need to exhibit a sustained and controlled release in order to decrease the number of doses and maintain optimum dosage levels. The rate at which the vector degrades determines the release kinetics and duration of the release. Sustained DNA release can prolong exogenous gene expression, thereby reducing the need for repeated dosing, which is a significant advantage for long-term gene therapy. Previous studies used hydrogels, polymer matrices, and microspheres to obtain a sustained controlled release. Microspheres formulated from hyaluronan exhibit a sustained release of pDNA over a couple of months, which is desirable for long term therapies. However, aggressive short term gene deliveries may be necessary for suicide gene therapy related to cancer treatment. Thus, more aggressive therapies would benefit from a polymer vector that degrades and produces a sustained release over several days.
The formulation of a non-viral vector with L-tyrosine polyphosphate (LTP) results in a vector that exhibits a sustained release over 7 days. LTP would be an ideal gene-vector for short term therapies. LTP is a biodegradable peptide polyphosphate that is synthesized from the natural amino acid L-tyrosine. LTP hydrolytically biodegrades at the phosphoester linkage and enzymatically degrades at the peptide linkage in the polymer backbone into L-tyrosine based derivatives and hence is suitable for biomaterial applications. The degradation products are nontoxic phosphates and L-tyrosine. Furthermore, the degradation products of LTP have negligible effect on local pH, which is unlike other biomaterials such as poly[DL-lactide-co-glycolide] (PLGA). L-tyrosine polyphosphate is soluble in a variety of common organic solvents and thus can therefore be processed into microsphere or nanosphere formulations.
wherein the above formula represents the chemical structure of L-tyrosine polyphosphate and where (1) through (4) represent the degradation sites thereof, where (1) is the backbone phosphoester bond—hydrolysis; (2) is the pendant phosphoester bond—hydrolysis; (3) is the pendant alkyl (hexyl) ester bond—hydrolysis; and (4) is the backbone amide (peptide) bond—enzymolysis and where x is an integer in the range of about 10 to about 80, or an integer from about 15 to about 75, or an integer from about 20 to about 70, or an integer from about 25 to about 60, or an integer from about 30 to about 50, or even an integer from about 35 to about 45. Here, as well elsewhere in the specification and claims, individual range limits can be combined to form additional non-disclosed ranges.
In another embodiment, the number of repeating units, x, in the above formula are selected so that the molecular weigh of the above L-tyrosine polyphosphate before degradation is approximately in the range of about 5,000 Daltons (Da) to about 40,000 Da, or from about 7,500 Da to about 30,000 Da, or from about 10,000 Da to about 25,000 Da, or even from about 15,000 Da to about 20,000 Da. Here, as well elsewhere in the specification and claims, individual range limits can be combined to form additional non-disclosed ranges. In another embodiment, the number of repeating units, x, is selected so that the molecular weight of the above L-tyrosine polyphosphate is about 11,000 Da.
Biodegradable nanospheres hold a unique advantage over the other non-viral vectors, since they are comparable in scale to viruses. Viruses range in size from tens to hundreds of nanometers. Previous studies show that particles with radii smaller than 50 nm exhibit significantly greater uptake by endocytosis or pinocytosis compared to particles larger than 50 nm with an optimal size around 25 nm. Some cell receptors can facilitate vector uptake into the cytoplasm directly across the plasma membrane, but the most common route for receptor-mediated uptake of macromolecular moieties is by endocytosis. Thus, endocytosis is an attractive mechanism for targeted gene delivery, which can be achieved by nanospheres. Also similar to viruses, once nanospheres are endocytosed they can provide an intracellular pDNA delivery that will avoid enzyme degradation in the circulation.
Nanospheres Encapsulating pDNA:
The most common method for encapsulating proteins or plasmids in nanospheres is an emulsion created by sonication (
Role of Chitosan Grafted with Polyethylene Glycol in Nanospheres:
Blending chitosan grafted with polyethylene glycol (PEG-g-CHN) into the nanospheres not only stabilizes the emulsion in order to increase nanosphere yield, but also enhances the biocompatibility. Several studies show PEG-g-CHN forms complexes with DNA and can be used a gene vector. Chitosan itself is shown to form complexes with pDNA and improve transfection efficiency. Furthermore, chitosan is approved by the FDA as a food additive and considered to be nontoxic. However, chitosan is limited as a gene vector since the crystalline structure of chitosan with its intra- and inter-molecular hydrogen bonds inhibits its solubility in organic solvents or aqueous solutions at physiological pH. By grafting polyethylene glycol (PEG) to chitosan through hydrogen bonding, an amphiphilic polymer is formed that is soluble in both dimethyl sulphoxide (DMSO) and acidic aqueous solutions. When formulating nanospheres with PEG-g-CHN (
The PEG-g-CHN, the generic structure of which is illustrated above, was purchased from CarboMer, Inc (Catalog No. 7-00105).
Incorporating the cationic polymer, linear polyethylenimine (LPEI), in the nanosphere formulation serves two major purposes. First, LPEI condenses pDNA, which prevents shearing of the pDNA during sonication. This prevention of shearing has been verified by preliminary research that revealed sonicated complexed pDNA with LPEI to be bioactive and intact. Condensation occurs due to the charge attractions between the positively charged LPEI and negatively charged pDNA. The N/P ratio is a measure of the ionic balance of the pDNA-LPEI complexes. The positive charge of LPEI originates from the nitrogen of the repeat unit of LPEI, NHCH2CH2, which has a molecular weight of 43 g/mol. The negative charge in the plasmid DNA backbone arises from the phosphate group of the deoxyribose nucleotides. The average molecular weight of the nucleotides is assumed to be 330 g/mol. Hence, a complex of a 1 mg of pDNA to 1 mg of LPEI is an N/P ratio of 7.7. Fortunately, pDNA-LPEI complexes are hydrophilic, so thermodynamics will favor their encapsulation in the inner water phase of the nanospheres during emulsification (
The second purpose of LPEI is its dramatic increase of vector transfection efficiency. Direct release of non-complexed pDNA in extracellular fluid or cytoplasm has poor transfection efficiency due to its inability to bind and pass through cellular membranes, escape entrapment in endosomes, and lysosomal degradation. The cellular membrane, like pDNA, has a negative charge. Thus, pDNA will repel from cellular membranes and is not likely to be endocytosed. Complexing the pDNA with positive cationic polymers like branched polyethylenimine (BPEI) and LPEI will help neutralize its charge. Both BPEI and LPEI condense DNA into complexes that are 50 to 100 nm in diameter, which is a particle size that can be endocytosed. These complexes are formed in low ionic strength solutions in the attempt to control the overall size of the complexes. Smaller complexes appear to have higher transfection efficiency and less toxicity than larger complexes. Numerous studies reveal that LPEI 25 kDa has a higher transfection efficiency and lower cytotoxicity than BPEI.
In the above formula, the number of repeating units, n, is selected so that the molecular weight of the above linear polyethylenimine composition is in the range of about 5,000 Da to about 50,000 Da, or from about 10,000 Da to about 40,000 Da, or from about 15,000 Da to about 30,000 Da, or even from about 20,000 Da to about 25,000 Da. Here, as well elsewhere in the specification and claims, individual range limits can be combined to form additional non-disclosed ranges. In another embodiment, n is selected so that the molecular weight of the above linear polyethylenimine composition is about 25,000 Da.
The exact mechanism of transfection enhancement by condensing pDNA with LPEI is unknown. However, LPEI is suspected to be able to escape entrapment by endosomes and eventual lysosome degradation. On the other hand, pDNA-LPEI is hypothesized to escape from endosomal entrapment by the “proton sponge theory” (
Successful gene therapy with a non-viral vector can be accomplished by overcoming the barriers such as: passing through the cell membrane, escaping from endosomes, protecting the DNA from enzyme degradation and shearing, and efficient transfection. These barriers will be overcome by nanospheres formulated from a polymer blend of L-tyrosine polyphosphate (LTP), polyethylene glycol grafted to chitosan (PEG-g-CHN), and plasmid DNA (pDNA) complexed with linear polyethylenimine (LPEI).
Agarose Gel Electrophoresis Assay of Sonicated pDNA-LPEI Complex:
Sonication during the emulsion step of nanosphere synthesis was known to shear and destroy pDNA. Therefore, plasmid DNA (pDNA) needed to be condensed with PEG-g-CHN, LPEI, or BPEI. To determine whether complexing pDNA with LPEI protected the pDNA from shearing during the sonication of nanosphere synthesis, an agrose gel electrophoresis assay was performed. First, all water used in the following experiments was distilled and de-ionized (Barnstead NanoPure II) and autoclaved (American Standard 25X-1) to inactivate DNAase. Next, PEF1-V5 (Invitrogen) plasmid DNA was propagated using a QIAGEN plasmid purification kit. LPEI (PolyScience Inc.) with a molecular weight of 25,000 Daltons was dissolved in dH2O at 70° C. at a concentration of 1 mg/ml. Then, 1:1 mass ratio of pDNA-LPEI (20 μg/ml pDNA and 20 μg/ml LPEI) samples were condensed for 45 minutes at 37° C. in 500 μL of autoclaved distilled and de-ionized H2O (dH2O). These samples were prepared in triplicate for both 30 second and 1 minute sonication times. Next, the sonicator tip (Branson 102C CE) was placed in the pDNA-LPEI samples and sonicated for either 30 seconds or 1 minute. Afterwards, 30 μL was taken from each sonicated sample and mixed with 6 μL of 6× dye (Sigma-Aldrich) then loaded into a 0.7% agarose gel containing ethidium bromide (0.5 μg/ml, Fisher Scientific).
Transfection Efficiency and Cytotoxicity of pDNA-Polymer Complexes:
Complexing pDNA with LPEI and BPEI have been shown to greatly enhance cellular transfection; however they were toxic at high dosages. Previous studies have shown that transfection increased with increasing the N/P ratio between pDNA and BPEI or LPEI. Therefore, the optimization of transfection efficiency with low toxicity was necessary. In order to compare transfection efficiency and cell viability of various sonicated and un-sonicated pDNA-polymer complexes, an X-Gal transfection assay was performed. First, PEG-g-CHN (CarboMer Inc.) with 80% acetylation was dissolved at a concentration of 3.33 mg/ml in 0.1N acetic acid for 48 hours at 37° C. under rotation. LPEI (PolyScience Inc.) with a molecular weight of 25,000 Daltons was dissolved in dH2O at 70° C. for 15 minutes at a concentration of 1 mg/ml. BPEI (PolyScience Inc.) with a molecular weight of 50,000 Daltons was prepared as a 30% aqueous solution. Then, pDNA-LPEI mass ratios of 1:1, 1:2, 1:4 and 1:8; pDNA-BPEI mass ratios of 1:1, 1:2, and 1:8; pDNA-PEG-g-CHN mass ratios of 1:1 and 1:10 were condensed for 45 minutes at 37° C. in 500 μL of dH2O with a pDNA concentration of 20 μg/ml. Next, primary human dermal fibroblasts (a gift from Judy Fulton at the Kenneth Calhoun Research Center, Akron General Medical Center) were seeded onto well tissue culture plates at a density of 25,333 cells/well and maintained overnight at 37° C. with fibroblast feeding media (90% Dulbecco's Modified Eagle Medium and 10% Fetal Calf Serum containing 1% Antimycotic). The next day, the fibroblast feeding media was replaced.
Next, 200 μL samples (4 μg of pDNA per sample) of pDNA-LPEI complex and of sonicated pDNA-LPEI complex were added to each well. Stock pDNA and TE buffer were used as negative controls. The positive controls were pDNA conjugated FuGENE 6 (Roche, Indianapolis, Ind.), a commercial transfection reagent. FuGENE 6 was prepared by incubating 4 μg of pDNA (40 μL) with 12 μL of FuGENE 6 and 148 μL of DMEM for 15 minutes. The cells were incubated for 72 hours at 37° C. Next, the cells were washed with phosphate buffer saline (PBS), fixed with 1% formaldehyde and an X-Gal staining assay was used to determine transfection. Five random photos were taken. The cells that appeared blue were successfully transfected and were expressing the β-gal enzyme. Transfection percentage was calculated by dividing the number of transfected (blue) cells by the total number of cells.
Nanospheres formulated with LTP, PEG-g-CHN, and pDNA-LPEI were prepared using an emulsion of water and oil by sonication and solvent evaporation technique (See Table 1 below). For each nanosphere formulation, PEG-g-CHN was dissolved at a 3.33 mg/ml concentration in 0.1N acetic acid for 48 hours at 37° C. LTP was synthesized according to the protocol established by Gupta and Lopina. LTP was dissolved in chloroform at a concentration of 100 mg/ml. Next, a 5% polyvinyl pyrrolidone (PVP) in dH2O was prepared. LPEI was dissolved in dH2O for 15 minutes at 70° C. at 3, 10 or 15 mg/ml. Then, for pDNA-LPEI complex loaded nanospheres, the pDNA and LPEI were complexed in dH2O for 45 minutes at concentrations of 0.3, 1.0, or 3.0 mg/ml each. Next, nanosphere formulations shown in below were emulsified by a sonicator (Branson 102C CE) for 1 minute. Nanosphere synthesis was performed in 6 replicates. A nanosphere batch with 10% loaded pDNA complexed with an equal amount of LPEI was also formulated (Table 2). Blank nanospheres and blank PLGA nanospheres were formulated as negative controls (Table 2). An additional batch of nanospheres was produced using a water-in-oil-in-water emulsion formed by an impeller (Yamato Lab-Stirrer LR400D).
Next, the chloroform was allowed to evaporate for 5 hours while the emulsion was gently stirred. The nanospheres were then collected by centrifugation at 15,000×g for 15 minutes. Afterward, the nanospheres were washed 3 times by centrifugation at 15,000×g for 15 minutes with autoclaved dH2O. The nanospheres were then shell frozen in 10 ml of dH2O, and were placed in a lyophilizer (Labconco Freezone 4.5) for 72 hours. Finally, the lyophilized nanospheres were stored in a desiccator.
Characterize the Size, Shape, Morphology, Degradation, and Cytotoxicity of pDNA-LPEI Loaded Nanospheres:
Rational: Intracellular delivery of pDNA to the nucleus should increase transfection efficiency by avoiding enzyme degradation in the circulation. The nanospheres must be endocytosed to accomplish intracellular delivery. In order to be endocytosed, the nanospheres must mimic the nanoscale of a virus. Therefore, verification was necessary to show that 1% loading of complexed pDNA in the nanospheres did not affect their size, shape, and morphology compared to blank nanospheres in the preliminary research. Furthermore, measuring nanosphere diameter as it degrades, characterized the degradation profile. In addition, fibroblasts exposed to nanospheres must have comparable cell viability to fibroblasts exposed to PLGA nanospheres and unexposed fibroblasts. The hypothesis was that SEM and laser light scattering would show that 1% complexed pDNA loaded nanospheres were comparable in size, shape, and morphology to blank nanospheres in the preliminary research. Furthermore, laser light scattering would show that nanospheres were completely degraded after 7 days based on previous research. Live/dead cell assay would show that fibroblasts exposed to 1% complexed pDNA nanospheres were comparable cell viability to unexposed fibroblasts.
Scanning Electron Microscopy of Nanospheres:
Scanning electron microscopy (SEM, Hitachi S2150) was used in order to qualitatively compare the size, shape, and morphology of 1% complexed pDNA loaded nanospheres to the blank and PLGA nanospheres. First, 1 mg of nanospheres was suspended in 1 ml of distilled and de-ionized H2O. Then, 200 μL of the suspended microspheres were pipetted onto a stub, dehydrated, sputter coated with silver/palladium, and examined. Blank nanospheres, 1% non-complexed nanospheres, 10% complexed pDNA nanospheres, impeller formed 1% complexed pDNA nanospheres, and PLGA nanospheres were used as controls.
Laser Light Scattering of Nanospheres:
Dynamic laser light scattering was used as an additional method for comparing the size of the 1% complexed pDNA loaded nanospheres to the blank nanospheres. The nanosphere sample was prepared by suspending 1 mg of nanospheres in 10 ml of PBS that had been passed through a 0.2 μm filter. The suspended nanospheres were centrifuged for 10 seconds at 1000×g to remove any large aggregates. Then, the sample was decanted into a glass scintillation vial. A dynamic laser light scattering system (Brookhaven Instruments BI-200SM) calculated the nanosphere diameter by the Regularized Non-negatively Constrained Least Squares (CONTIN) method. The range of nanosphere size was reported as differential distribution values. The differential distribution value varied from 0 to 100, not percent, just 100. The highest peak or modal value was assigned to the number 100. For example, if the diameter of 150 nm had the differential distribution value of 37 and at the diameter of 200 nm the differential distribution value was 74, then, the distribution had twice the amount at 200 nm than it did at 150 nm. The differential distribution values were the relative amount at the corresponding diameter. Blank nanospheres, 1% non-complexed nanospheres, 10% complexed pDNA nanospheres, impeller formed 1% and blank PLGA nanospheres will be used controls.
Degradation of Nanospheres:
To quantify the release duration and the degradation of the nanospheres in vitro, laser light scattering was utilized. Light scattering samples and the procedure was performed according to the procedure described herein. Then, the nanospheres were incubated at 37° C. and slightly shaken for 11 days. On days 0, 1, 2, 3, 4, 7, and 11, laser light scattering was performed in order to measure nanosphere diameter. The mean diameter of nanospheres was calculated by the Brookhaven software and reported for each day. Blank nanospheres, 1% non-complexed nanospheres, and blank PLGA nanospheres were used controls.
Cell Viability after Exposure to Nanospheres:
The cell viability of primary human dermal fibroblasts after exposure to 1% complexed pDNA nanospheres was determined using a live/dead cell assay (Invitrogen). First, primary human dermal fibroblasts (a gift from Judy Fulton at the Kenneth Calhoun Research Center, Akron General Medical Center) were seeded onto 24 well tissue culture plates at a density of 25,333 cells/well and maintained overnight at 37° C. with fibroblast feeding media (90% Dulbecco's Modified Eagle Medium and 10% Fetal Calf Serum containing 1% Antimycotic). The next day, the fibroblast feeding media was replaced. Fibroblasts were exposed to 400 μg of nanospheres. After 1, 3, 7, and 11 days a live/dead cell assay was performed according to the manufacturer's instructions. Blank nanospheres, pDNA-LPEI, pDNA-FuGENE 6, 10% pDNA nanospheres, impeller formed 1% complexed pDNA nanospheres, and blank PLGA nanospheres were used controls.
Quantify and Characterize Nanosphere Loading and Release of pDNA-LPEI:
Rationale: A sustained release of complexed pDNA would decrease the number of dosages. In order to treat short term gene therapies, a sustained release of intact complexed pDNA for approximately 7 days was desired. Therefore, the release of complexed pDNA over 7 days was characterized, quantified, and structurally examined. The hypothesis was that a sustained release would be observed over a course of at least 3 days based off of preliminary research. Furthermore, a significant difference was expected between the transfection efficiency of the release samples and of the negative controls based on preliminary results. AFM was hypothesized to reveal intact pDNA-LPEI complexes released from the 1% complexed pDNA nanospheres. The released pDNA-LPEI complexes would not be significantly different in size to the stock pDNA-LPEI complex.
Loading Efficiency of pDNA in Nanospheres:
The loading efficiency of the various nanosphere formulations was performed using a PicoGreen® (Molecular Probes) fluorescence assay. The loading of all formulations of pDNA nanospheres was determined by dissolving 2 mg of nanospheres in 0.2 ml of chloroform for 1 hour at 37° C. Then, an equal volume of autoclaved TE buffer was added and lightly shaken for 2 minutes. The phase separation between the chloroform and the TE was allowed to form after 30 minutes. This mixture was then centrifuged for 5 seconds at 10,000×g. Next, 200 μL of TE supernatant was sampled. The amount of pDNA was determined with a PicoGreen® (Molecular Probes) fluorescence assay according to manufacture's instructions.
Agrose Gel Electrophoresis of pDNA Released from Nanospheres:
The release of pDNA-LPEI from 1% complexed pDNA loaded nanospheres was characterized. One batch each of impeller formed 1% complexed pDNA nanospheres and 10% complexed pDNA nanospheres was also analyzed. Blank and 1% non-complexed nanospheres were used as controls. First, 2 mg of each nanosphere formulation was suspended in 500 μL of TE buffer and incubated at 37° C. under constant rotation. Next, after 30 minutes, 1, 3, 6, 12 hours, 1, 2, 3, 4, 5, 6, 7 days, the nanosphere suspensions were centrifuged at 10,000×g, 450 μL of the supernatant was collected and replaced with an equal volume of fresh TE buffer. Furthermore, the release samples were lyophilized and re-suspended in 100 μL of TE buffer. The structural integrity of the pDNA and pDNA-LPEI released from the 1% non-complexed pDNA loaded nanospheres were analyzed by agrose gel electrophoresis. First, 30 μL samples from each release time point and nanosphere formulation were mixed with 6 μL of 6× loading dye and loaded into a 0.8% agrose gel containing ethidium bromide.
Release Profile of Nanospheres Based from Transfection:
The quantitation of the bioactivity of pDNA-LPEI released from the nanospheres was determined with an X-Gal transfection assay using primary human dermal fibroblasts. The cells were maintained according to the protocol in Section 3.2.4 of this thesis. Next, 40 μL release samples was added to the feeding medium. Control fibroblasts were transfected with stock pDNA using a mixture of 200 ng (2 μL), 1.9 μL of FuGENE 6, and 96.1 μL of DMEM. Stock pDNA, release from blank nanospheres, 1% non-complexed pDNA loaded nanospheres, and TE buffer will be used as negative controls. Next, the cells were washed with phosphate buffer saline (PBS), fixed with 1% formaldehyde and an X-Gal staining assay was used to determine transfection. Five random photos were taken. The cells that appeared blue were successfully transfected and were expressing the β-gal enzyme. Transfection percentage was calculated by dividing the number of transfected (blue) cells by the total number of cells.
Atomic Force Microscopy of Released pDNA-LPEI:
The physical structure of the released pDNA-LPEI complexes was characterized using atomic force microscopy (AFM, Vecco Nanoscope III). First, stock samples of pDNA and pDNA-LPEI at concentrations of 20 μg/ml were analyzed in order to obtain a benchmark visual. Samples were prepared by pipetting 20 μL of the release onto a silicon wafer (donated by Bi-min Zhang Newby, Department of Chemical Engineering, University of Akron), dehydrated, and visualized using AFM. Blank nanospheres, stock pDNA, and stock pDNALPEI complex will be used controls. The structure of the released pDNA-LPEI complex was compared to the structure of stock pDNA-LPEI complex.
Verify Nanospheres are Taken up by Cells and Achieve Higher Transfection Efficiency as Compared to pDNA Alone:
Rationale: An overall enhancement of transfection through a sustained release of pDNA-LPEI complexes from endocytosed nanospheres as compared to pDNA alone needs to be verified. Therefore, endocytosis of nanospheres and the increase in transfection efficiency was verified. The hypothesis was that cellular uptake of the FITC labeled nanospheres will be visualized and verified using confocal microscopy. In addition, a significant difference in the transfection efficiency was hypothesized to exist between the control cells exposed to 4 μg of pDNA and the cells exposed to 400 μg of either 1% or 10% complexed pDNA loaded nanospheres.
Cellular Uptake of Nanospheres:
In order to determine if the LTP/PEG-g-CHN/LPEI nanospheres were taken up by endocytosis, primary human fibroblasts were exposed to FITC labeled nanospheres and visualized with confocal microscopy (Olympus Fluoview). First, FITC loaded (1%) nanospheres were produced according to the aforementioned nanosphere synthesis procedure in Section 3.2, except 0.3 mg/ml FITC solution in 1 ml of DMSO was used in place of 1 ml of dH2O, Next, human dermal fibroblasts were seeded onto sterile German glass cell culture cover slips (Fisher) in 24 well tissue culture plates at a density of 25,333 cells/well and maintained overnight at 37° C. with fibroblast feeding media. The next day, the fibroblast feeding media was replaced. Then, 1 mg of FITC labeled nanospheres was suspended in 1 ml of dH2O, Next, 80 μL of the suspended nanospheres were added to the feeding medium of each well. Human dermal fibroblast cells without any exposure to FITC loaded nanospheres were seeded onto German glass cover slips were used as negative controls. After 24 hours of incubation, the fibroblast cells were washed with PBS and fixed with 1% formaldehyde in PBS for 10 minutes. Next, the cells were washed with PBS, and then 2.5 μL of 6.6 μM Rhodamine Phalloidin stock solution (Molecular Probes) was diluted to 100 μL in PBS and added onto each cover slip. After 20 minute incubation at room temperature, the cells were washed with PBS. The cover slips were then mounted to glass slides with Vectashield mounting media containing DAPI (Vector laboratories). Excess mounting media was removed; the cover slips were sealed and stored at 4° C. Next, FITC labeled nanosphere cellular uptake was visualized first using fluorescent microscopy (Axiovert 200, Carl Zeiss), and then with confocal microscopy (Olympus Fluoview) at NEOUCOM with 2 channels of fluorescence (FITC and Rhodamine), and photographed with a phototube. Confocal microscopy assistance was provided by Jeanette G. Killius at NEOUCOM.
Transfection Efficiency of Nanospheres:
To qualitatively examine the transfection efficiency of both 1% and 10% complexed pDNA loaded nanospheres, an X-Gal transfection assay was performed on primary human fibroblasts with the direct addition of nanospheres. First, human fibroblast cells were seeded onto 24 well tissue culture plates at a density of 25,333 cells/well and maintained overnight at 37° C. with fibroblast feeding media. The next day, the fibroblast feeding media was replaced. Next, 2 mg of nanospheres were suspended in 500 μL of feeding medium. Then, 100 μL of the suspended nanospheres were added to the cells. Control fibroblasts were transfected with stock pDNA using a mixture of 200 ng (2 μL), 1.9 μL of FuGENE 6, and 96.1 μL of DMEM. After 3, 5, 7, 9, and 11 days of incubation, the fibroblast cells were fixed with 1% formaldehyde in PBS for 10 minutes. Next, an X-Gal transfection assay was performed according to the manufacturer's instructions. For each transfection result (n=3 for each time point), three random fields were selected using a microscope (Axiovert 200, Carl Zeiss) with a 20× magnification lens, and bright field images were captured using a Cannon Power Shot G5 camera. The cells that appeared blue were successfully transfected and were expressing the β-gal enzyme. Blank nanospheres, 4 μg of complexed pDNA, pDNA with FuGENE 6, blank TE buffer, and 4 μg of pDNA were used controls.
Statistics:
All quantitative studies were performed in 6 replicates determined by power analysis with α=0.05. The nonparametric Kruskal-Wallis analysis of variance was used to determine statistical differences within each sample group. All results were considered significant when p≦0.05. If no significant differences were found within a sample group, then the sample was considered normally distributed. Tukey's analysis of variance was then performed among the normally distributed sample groups. All results were considered significant if p≦0.05.
Preliminary Results: Agarose Gel Electrophoresis Assay of Sonicated pDNA-LPEI Complex:
Studies in the past have shown that pDNA complexed with polycationic polymers results in a high molecular weight band in the loading wells of the gel. These bands were visible in the gel for the sonicated samples 1:1 pDNA-LPEI and for stock 1:1 pDNA-LPEI (
Transfection Efficiency and Cytotoxicity of pDNA-Polymer Complexes:
The in vivo addition of complexes of 1:1 pDNA-LPEI, 1:1 pDNA-PEG-g-CHN, and 1:10 pDNA-PEG-g-CHN yielded high cell viability, which was comparable to blank cells and cells transfected only with pDNA as shown in
Fibroblasts exposed to 1:1 pDNA-LPEI (4 μg of LPEI) produced the highest transfection percentage of approximately 30% (
Scanning Electron Microscopy of Nanospheres:
Scanning electron microscopy (SEM) is utilized to examine the nanospheres' morphology, size, and shape. The images obtained by the SEM reveal a smooth surface morphology of all nanosphere formulations (
Laser Light Scattering of Nanospheres:
Dynamic laser light scattering is used as an additional method to quantitatively measure the nanosphere diameter range. The frequencies of the nanosphere diameters are reported as differential distribution values. Laser light scattering measures the blank nanosphere diameter range to be between 156 to 562 nm (
Degradation of Nanospheres:
Dynamic laser light scattering is further utilized to characterize the degradation of the nanospheres. The degradation is equated as the decrease in mean diameter of the nanospheres after 7 days. Blank nanospheres are completely degraded in PBS at 37° C. after 7 days (
Cell Viability after Exposure to Nanospheres:
The viability of human dermal fibroblasts 24 hours after exposure to 1% pDNA nanospheres is determined using a LIVE/DEAD Cell Assay. In the LIVE/DEAD cell assay, metabolically active cells reduce C-resazurin to red fluorescent C-resorufin and dead or dying cells fluoresce green, since their plasma membranes are compromised and are permeable to the nucleic stain SYTOX. Nanospheres, which fluoresced green with the same filter as dead cell nuclei, were distinguished by their smaller and more spherical shape as opposed to the larger bean shaped nuclei. In
Cell Viability=[(red cells)/(red cells+green cells)]×100
The addition of the positive control buffer, 40 μL of TE buffer and 160 μL of dH2O, to fibroblasts results in a 98%±1% cell viability after 24 hours (
Statistical analysis is performed to determine significant differences among the gene vectors and controls. First, a Kruskal Wallis nonparametric test of variance demonstrates that there is no significant difference within each group of gene vectors, which shows that the samples are normally distributed. Therefore, Tukey's analysis of variance is performed among the groups of gene vectors, since each group has a normally distributed sample. Tukey's comparison of means finds that after 1, 3, 7, and 11 days, the viability of 1% pDNA nanospheres is not significantly different (p>1.000) to TE buffer, PLGA nanospheres, or pDNA. After 1 day, only 10% pDNA nanosphere viability was found to be significantly different (p=0.0003) than TE buffer. After 3 and 7 days, TE Buffer viability is significantly different than pDNA-FuGENE 6 (p<0.0001) and 10% pDNA nanospheres (p=0.0047) and (p=0.0021) respectively. After 11 days, TE buffer is significantly different than blank nanospheres (p<0.0001), pDNA-FuGENE 6 (p<0.0001), 10% pDNA nanospheres (p<0.0001), and pDNA-LPEI (p=0.0006).
Loading of pDNA into Nanospheres:
The loading efficiency of pDNA into the nanospheres is determined using a PicoGreen® DNA quantitation assay. A decrease in fluorescence is observed when pDNA is complexed with LPEI. Therefore, a standard curve for emission fluorescence and concentration is made using titrations of the pDNA-LPEI complexes. The loading of pDNA-LPEI is determined from the standard curve. Loading efficiency is defined by the following equation:
Loading Efficiency=[(measured amount of pDNA from nanospheres)/(amount of pDNA in nanospheres)]×100
The loading efficiencies for the sonicator formed 1% and 10% pDNA nanospheres are 40%±3% and 13%±1% respectively (
Statistical analysis is performed to determine significant differences among the loading efficiencies. Initially, a Kruskal Wallis nonparametric test of variance demonstrates that there is no significant difference within each group of nanospheres, which shows that the samples are normally distributed. Therefore, Tukey's analysis of variance is performed among the groups of nanospheres, since each nanosphere has a normally distributed sample. Tukey's test shows that the loading efficiencies of all nanosphere formulations are significantly different from each other (p<0.0001).
Agrose Gel Electrophoresis of pDNA Released from Nanospheres:
In order to qualitatively characterize the release of nanospheres, an agrose gel electrophoresis is performed. In gel electrophoresis, pDNA migrates from a well through the gel due to a charge gradient. When pDNA is complexed with cationic polymer LPEI at a 1:1 mass ratio (7.7 N/P ratio), it loses its negative charge and does not migrate through the gel. Thus, bands that remain in the wells are determined to be pDNA-LPEI complexes. Agrose gel electrophoresis of blank nanospheres results in low molecular weight bands (fast migrating particles) below the 1 kb DNA ladder (
Gel electrophoresis of the 1% complexed pDNA nanosphere release is sustained over 7 days (
The electrophoresis gel of release from 10% complexed pDNA nanospheres resembles the release of the 1% complexed pDNA nanospheres, but with greater intensity of pDNA and pDNA-LPEI (
The impeller formed 1% complexed pDNA nanospheres yields a similar release to the sonicator formed nanospheres. However, the impeller nanospheres demonstrate their largest release after 0.5 and 1.5 hrs of pDNA-LPEI, sheared pDNA, relaxed and super coiled dimmer pDNA, and degradation products (
The release of the pDNA from the 1% non-complexed pDNA nanospheres was visible in the agrose gel electrophoresis assay for the first 2 days of the release only as shown in
Release Profile of Nanospheres Based from Transfection:
The release profile of various nanosphere formulations is quantified using transfection percentages obtained from the release samples. However, insufficient transfection is obtained from the 1% pDNA nanosphere release samples. Transfection is only obtained from the day 2 release samples of 1% pDNA nanospheres (
Cellular Uptake of Nanospheres:
Cellular uptake of nanospheres is confirmed with confocal microscopy of human dermal fibroblasts exposed to FITC loaded nanospheres. First, a preliminary study is performed using fluorescent microscopy with 3 channels of fluorescence (Axiovert 200, Carl Zeiss). The fibroblasts' nuclei are stained with Hoechst nuclear stain and fluoresce blue. The cytoskeleton is stained with rhodamine phalloidin and fluoresces red. These cellular stains can be seen for both control fibroblasts (
Therefore, a 3-dimensional analysis of the uptake is achieved using confocal microscopy on fibroblasts exposed to 100 μg of FITC loaded nanospheres. Confocal microscopy produced 0.5 μm slices of the fibroblast samples. These slices revealed nanospheres at various depths within the fibroblasts' cytoskeleton (
Transfection Efficiency of Nanospheres:
The controllable and sustained transfection from nanospheres is demonstrated by X-Gal staining of human dermal fibroblasts exposed to 400 μg of 1% complexed pDNA nanospheres. X-Gal staining is used to determine the percentage of cells transfected with pDNA expressing lacZ. The product of the lacZ gene, β-galactosidase, catalyzes the hydrolysis of X-gal, producing a blue color within the cell. Transfection percentage is used to determine the transfection efficiency of the nanospheres. The transfection percentage is calculated by the following equation:
Transfection Percentage=[(blue cells)/(total cells)]×100
Fibroblasts exposed to 4 μg of pDNA (200 μL of 20 μg/ml) after 3, 5, 7, 9 and 11 days demonstrate no transfection, which is comparable to buffers alone (
Unlike pDNA complexes with LPEI and FuGENE 6, nanospheres demonstrate a controllable and sustained transfection (
Statistical analysis is performed in order to establish significant differences in transfection efficiencies among the gene vectors. First, a Kruskal Wallis nonparametric test for variance demonstrates that there is no significant difference within each group of vectors, which shows that the vector samples are normally distributed. Then, Tukey's analysis of variance is performed among the groups of gene vectors, since each group has a normally distributed sample. On day 3, the transfection percentage for 1% pDNA nanospheres was found to be significantly different (p<0.0001) than pDNA-LPEI and pDNA-FuGENE 6. On day 5, the transfection percentage of 1% pDNA nanospheres was not found to be significantly different than either pDNA-LPEI (p=0.9989) or pDNAFuGENE 6 (p=0.9996). However on day 7, a significant difference exists between 1% pDNA nanospheres and pDNA-LPEI (p=0.0146) and pDNA-FuGENE 6 (p=0.0121). In addition, no significant differences were observed between transfection from nanospheres on days 7, 9, or 11 (p=0.9949, p=0.8292, p>1.00 respectively). No significant differences in transfection percentage were found between 1% pDNA nanospheres and pDNA-LPEI for days 5, 9, and 11 (p=0.9989, p=0.1769, and p=0.2863) and pDNA-FuGENE 6 for days 5, 9, and 11 (p=0.9996, p=0.3176, p=0.7329).
Gel Electrophoresis of Sonicated pDNA-LPEI Complex:
Gel electrophoresis demonstrates that complexing pDNA with a polymer such as LPEI prevents large scale degradation of pDNA during the sonication step of nanosphere production. The complexing of pDNA by LPEI produces a condensed and more structurally stable package than pDNA alone. This smaller and more robust package can withstand the large amounts of energy and forces present in the emulsion created by sonication. Sonicating pDNA alone, degrades pDNA, and renders it inactive for transfection.
Transfection Efficiency and Cell Viability of pDNA-Polymer Complexes:
The complex of pDNA and LPEI is the most efficient pDNA-polymer complex at transfecting cells while maintaining acceptable cell viability. LPEI has better transfection efficiency than FuGENE 6, BPEI, and PEG-g-CHN, which is believed to be due to its ability to escape endosomes. In addition, LPEI demorstrates higher cell viability than FuGENE 6 and BPEI. The optimum mass ratio of pDNA to LPEI is 1 to 1, which corresponds to a 7.7 N/P. The 1:1 mass ratio of pDNA and LPEI produces neutrally charged complexes that have the highest transfection efficiency compared to other mass ratios, which is a result of the complete neutrality of charge. Future studies must explore the structure and size of the pDNA-LPEI complex, in order to better understand its mechanism for transfection.
Nanospheres can be synthesized from L-tyrosine polyphosphate, PEG-g-CHN, and pDNA-LPEI using both the sonication and impeller methods to create water and oil emulsions. These nanosphere formulations represent the first attempt to achieve a controlled and sustained intracellular delivery of pDNA for gene therapy. Both the sonication and impeller method are effective at producing pDNA loaded nanospheres.
Characterize the Size, Shape, Morphology, Degradation, and Cytotoxicity of pDNA-LPEI Loaded Nanospheres:
SEM of Nanospheres:
The nanospheres' shape, surface morphology, and size play a major role in biocompatibility and the ability to be internalized. Spherical and smooth particles demonstrate favorable transport in circulation and biological systems. Meanwhile, irregular and rough particles pose problems when navigating through microcirculation. Fibrous particles have been shown to stress cells and elicit immune responses. Previous studies show that cellular internalization of particles is size dependent. Eukaryotic cells can internalize nanoparticles with diameters ranging from 50 nm to 1 μm. Therefore, nanospheres must be produced with diameters smaller than 1 μm for intracellular delivery of genes. Scanning electron microscopy (SEM) reveals that our 1% pDNA nanospheres are spherical, smooth, and range in diameter between 200 to 700 nm (
Laser Light Scattering of Nanospheres:
The results from the dynamic laser light scattering reaffirm nanosphere diameter range found with SEM. Laser light scattering shows that all the nanospheres formulations have a near normal distribution of diameters. The diameter distribution of impeller formed nanospheres is smaller and narrower (100 nm to 500 nm) than the sonicator formed, which could be attributed to greater energy created in the impeller emulsion. Furthermore, the impeller formed nanospheres are created in a water-in-oil-in-water emulsion, which may have greater emulsion stability than a sonication emulsion. Therefore, all pDNA nanosphere formulations are favorable in size for cellular internalization.
Aggregation of nanospheres is also observed using laser light scattering. The nanosphere aggregates appear as, particles with 1 to 10 μm diameters (
Degradation of Nanospheres:
Nanosphere degradation based off changes in mean diameter over time, shows that all pDNA nanosphere formulations are fully degraded after 7 days. This degradation profile is comparable to the 7 day degradation of LTP films incubated in PBS, which is expected since the nanospheres are about 90% LTP. All nanosphere formulations are approximately 75% degraded after 3 days (
The increase in mean nanosphere diameter from day 0 to day 1 with the sonicator formed pDNA nanospheres can be explained by increased aggregation (
Cell Viability after Exposure to Nanospheres:
The LIVE/DEAD cell assay demonstrates that 1% pDNA nanospheres have viabilities comparable to buffers, pDNA alone, and PLGA nanospheres. Other gene vectors such as LPEI and FuGENE 6 demonstrate increasing toxicity between 1 and 11 days of incubation with human dermal fibroblasts. The 1% pDNA nanospheres avoid these toxic effects by encapsulating the toxic LPEI and releasing it at a controlled and sustained rate. The nanospheres prevent the burst toxic effects of LPEI, which previous studies show to be toxic to cells at high concentrations. High cell viability is also due in part to the nontoxic polymers used to fabricate the nanospheres. LTP is a biocompatible polymer synthesized from the amino acid L-tyrosine and phosphate groups, which both are found naturally in the body. The hydrolytic degradation of LTP results in L-tyrosine and a phosphate that are both nontoxic. PEG and Chitosan have also been shown to be nontoxic. The 1% pDNA nanospheres are safe to use in vitro with fibroblasts at their effective concentrations, since they have comparable viabilities to buffers, pDNA, and PLGA nanospheres.
The LIVE/DEAD cell assay also demonstrates the degradation of the nanospheres as well. The fact that the nanospheres fluoresce green provides an opportunity to watch the nanospheres diminish in fluorescence as they degrade throughout the 11 days (
Quantify and Characterize Loading and Release pDNA-LPEI from Nanospheres:
Loading of pDNA-LPEI in Nanospheres:
The PicoGreen assay shows that the loading efficiency varies among the various pDNA nanosphere formulations. The 10% pDNA nanospheres have the lowest loading efficiency at 13%, which can be attributed to the pDNA-LPEI concentration exceeding the nanospheres maximum loading. Micro and nanoparticles have a maximum loading, which cannot be increased with additional loading materials. Excess non-encapsulated pDNA-LPEI is washed away during the nanosphere collection and washing steps. The 1% pDNA nanospheres have the next highest loading efficiency at 40%. Further investigation is needed to determine if a 1% pDNA-LPEI concentration is the optimum concentration for loading in this nanosphere formulation. The sonication method for nanosphere formation relies on the random encapsulation of hydrophilic pDNA-LPEI in the inner water phase, due to the thermodynamic favorability. However, low encapsulation can occur when the hydrophilic pDNA-LPEI is caught in the outer water phase, which is also thermodynamically favorable. The impeller formed 1% nanospheres have the highest loading at 89%. The impeller method achieves a higher loading efficiency due to the nature of its formation, which includes and an initial water-in-oil emulsion. Unlike the sonication method where pDNA-LPEI can collect randomly in the outer or inner water phase, the initial water-in-oil emulsion forces nearly all the pDNA-LPEI in the inner water phase. Thermodynamics favor the pDNA-LPEI to exist only in the water phase. A common factor in both sonication and impeller systems that leads to a decrease in loading is the destruction of pDNA-LPEI during the emulsion. The emulsion generates a large amount of energy that can shear and destroy pDNA, which is demonstrated in the release of non-complexed pDNA nanospheres (
Agrose Gel Electrophoresis of pDNA Released from Nanospheres:
Gel electrophoresis shows a sustained release of PDNA-LPEI complexes from nanospheres throughout 7 days. The electrophoresis gel of the 1% pDNA nanospheres shows the greatest release is found during the first 2 days, which corresponds to the nanosphere degradation observed in
The completely sheared pDNA found in the non-complexed pDNA nanosphere release further demonstrates the importance of complexing the pDNA before encapsulation in nanospheres. Encapsulating pDNA alone in nanospheres via the sonication method results in an ineffective gene vector, since the pDNA is no longer bioactive.
Release Profile of Nanospheres Based from Transfection:
Using transfection efficiency of nanosphere release in order to generate a release profile is unsuccessful, due to the inability of the release from 1% pDNA nanospheres to transfect cells. Only the release from day 2 of the 1% pDNA nanospheres achieved transfection. This lack of transfection could be attributed to loss of bioactivity of the pDNA during the experimental procedure of the release. The lyophilization and re-suspension of the release may destroy the bioactivity of the pDNA. In addition, the low transfection could be a result of the low loading efficiency found with the 1% pDNA nanosphere. Only 40% of the expected pDNA-LPEI is encapsulated in the nanospheres, which could lead to low transfection. This hypothesis is supported by the higher transfection obtained by the release from 10% pDNA nanospheres and impeller formed 1% nanospheres, which has higher loading than 1% pDNA nanospheres. There is more pDNA-LPEI present in these nanospheres, which leads to a greater release. Future release studies must be performed with a greater amount of 1% pDNA nanospheres in order to obtain transfection from the release. Other future studies could quantify the release of radioactively labeled pDNA-LPEI, since regulations and cost prevented such studies in this current research.
Verification that Nanospheres are Taken up by Cells and Achieve Higher Transfection Efficiency as Compared to pDNA Alone:
Cellular Uptake of Nanospheres:
Confocal fluorescent microscopy has verified the uptake of nanospheres by primary human dermal fibroblasts after 24 hours. Individual nanospheres can be seen appearing and disappearing within the cytoskeleton of the fibroblasts, which proves that they are inside the cell. In addition, nanospheres only require 24 hours to be taken up by the fibroblasts, which ensures that the nanospheres are not fully degraded before they can be internalized. The rapid uptake of these nanospheres is important for ensuring an internal release of pDNA-LPEI, which should improve transfection efficiency. The fluorescent images alone of fibroblasts with nanospheres only suggest an uptake of nanospheres. Although, the confocal images show that many of the nanospheres seen with the fibroblasts are likely inside the cell. Some nanospheres may also be still adhered to the membrane of the cell as well. The specific location of the nanospheres within the cell is unknown at this time. However, the nanospheres are hypothesized to exist within endosomes where they are being degraded. Studies by Leong show that internalized nanospheres are typically found in endosomes. Future studies are needed to verify the location of the nanospheres within endosomes. An assay for endosomal staining can demonstrate that the nanospheres are contained within endosomes. Furthermore, future studies can also verify how the nanospheres uptake the nanospheres. An enzymatic assay to determine receptor-mediated endocytosis must be performed to verify that endocytosis is indeed the method for nanosphere internalization.
Transfection Efficiency of Nanospheres:
X-Gal staining of human dermal fibroblasts exposed to 1% pDNA nanospheres shows a controlled and sustained transfection. The transfection is considered controlled, since transfection is not observed until 5 days after their exposure to cells. Unlike previously established gene vectors such as LPEI and FuGENE 6, there is a delay in transfection until day 5 when using the 1% pDNA nanospheres. This delay is a result of nanospheres taking time to reach the cell, be internalized, and degrade within the cell. Maximum transfection from the 1% pDNA nanospheres is reached on day 7 and is sustained through day 11. The fact that no significant differences were found between nanosphere induced transfection on days 7 through 11, demonstrates that transfection is sustained. The sustained transfection is achieved due to the nanosphere degradation time frame of 7 days (
The 1% pDNA nanospheres' controlled and sustained transfection is in stark contrast to the initial burst and decaying transfection of the LPEI and FuGENE 6. The established gene vectors LPEI and FuGENE 6 achieve very high transfection initially. However, LPEI and FuGENE's transfection diminishes between 3 to 11 days due to their inability to control or sustain their transfection. Cationic polymers and lipoplexes are easily cleared by cells and biological systems. Gene vectors such as LPEI must be incorporated into a degradable system in order to obtain a controlled or sustained transfection. The degradable nanospheres provide a means of controlling and sustaining transfection with LPEI.
Despite the transfection success of 1% pDNA nanospheres, poor results were found for 10% pDNA nanospheres and impeller formed nanospheres. Transfection could not be achieved using 10% pDNA nanospheres. Meanwhile, impeller formed 1% pDNA nanospheres demonstrate very low transfection, but still exhibit a controlled and sustained transfection. However, the sustained transfection obtained by impeller nanospheres is not significantly different from LPEI and FuGENE 6 on days 5 through 11. The low or lack of transfection is a likely a result of the size of the pDNALPEI complexes in the 10% pDNA nanospheres. Studies show that the smaller the pDNA-LPEI complex size, the greater the transfection. Furthermore, the studies show that the higher the concentration of pDNA and LPEI in solution, the larger the complexes that form. The concentration of both pDNA and LPEI is 0.3 mg/ml, 1 mg/ml, and 3 mg/ml when forming the complex for 1% pDNA nanospheres, 10% pDNA nanospheres, and impeller formed 1% pDNA nanospheres respectively. This increase in concentration likely increased the size of the pDNA-LPEI complex, which lowered the transfection. In addition, the cell viability studies show that the 10% pDNA nanospheres are more toxic to cells than the 1% pDNA nanospheres. An increase in cell toxicity leads to poor cell function and less gene expression.
Nanospheres formulated from a blend of LTP and PEG-g-CHN that encapsulate pDNA-LPEI can be used as a controllable and sustainable non-viral gene vector. An emulsion of water and oil produced by sonication and solvent evaporation creates the nanospheres and leads to the encapsulation of the pDNA-LPEI. This fabrication method produces nanospheres that are spherical, smooth, and approximately 100 to 700 nm in diameter. These nanospheres degrade in 7 days in PBS at 37° C. This degradation profile leads to a release of pDNA-LPEI over 7 days with most of the release occurring in the first 2 days. A formulation of 1% pDNA nanospheres exhibits higher cell viability than other established gene vectors such as LPEI and FuGENE 6. The cell viability of these nanospheres is comparable to TE buffer, pDNA, and PLGA nanospheres. The high viability of these nanospheres is due in part to the biocompatibility of the polymers used and the size of the nanospheres. The nanosphere size also provides a suitable scale for internalization by the cells. Uptake of the nanospheres by fibroblasts is achieved in 24 hours, which allows time for an intracellular delivery of pDNA-LPEI. This intracellular delivery leads to a controlled and sustained transfection of human dermal fibroblasts. These nanospheres achieve a controlled transfection by delaying prominent gene expression until 5 days after administration. Maximum transfection is reached on day 7 and sustained through day 11, which is unlike the initial burst transfection and then decay of gene vectors such as LPEI and FuGENE 6. Therefore, the nanospheres formulated from LTP, PEG-g-CHN, and pDNA-LPEI could be valuable vectors for intracellular delivery of therapeutic genes against diseases that require treatment for a couple weeks.
In another embodiment, the present invention relates to polymers that contain at least one amino acid in their backbone. In yet another embodiment, the present invention relates to polymers that contain at least one amino acid in their backbone thereby resulting in biodegradability, and in some embodiments, controlled biodegradability. In still another embodiment, the present invention relates to phosphate and/or urethane-based polymers that have at least one amino acid in their backbone thereby resulting in biodegradability. In still another embodiment, the present invention relates to L-tyrosine-based phosphate polymers and/or L-tyrosine-based urethane polymers for biomaterial applications.
The use of amino acids in the synthesis of polymers for different biomaterial applications is known (see, e.g., U.S. Pat. No. 6,221,997 which discloses pendant chain amino acid-containing polyurethanes). L-tyrosine has been extensively used for the synthesis of biocompatible and/or biodegradable polymers for different biomaterial applications with particular emphasis on tissue engineering. In particular, L-tyrosine-based pseudo-poly (amino acids) has been investigated for biomaterial applications with desaminotyrosyl hexyl ester (DTH) as the building unit for the polymers. DTH based polycarbonate, polyirnminocarbonate, polyphosphates and several other polymers are studied for biomaterial applications. However, the uses of these polymers are restricted due to several limitations regarding degradability, physical-chemical properties and processability as well.
Moreover, the difficulties in tuning the polymer structure and the related properties of the material have limited their chances of using these materials for wide range of applications.
Biocompatible polyurethanes are currently being investigated as an alternative for fabricating tissue engineering scaffolds. Several studies indicate that the ease of synthesizing and tuning the structure leads to wide range of properties that are pertinent to biomaterial applications. Polyurethanes are usually synthesized from three components, macrodiol polyol), diisocyanate, and diol or diamine based chain-extenders and has the general structure as shown below. This enables to constitute the soft segment and the hard segment of the polymer, which eventually can be exploited for various properties.
-M-(D(CD)n-M)m-
The above formula is a general schematic for the L-tyrosine-based polyurethane polymers where M=macrodiol, D=diisocyanate, and C=chain extender.
Amino acid based chain extenders are investigated for polyurethanes in very limited cases. Phenyl alanine based chain extender and lysine based isocyanate is used for the synthesis of polyurethanes.
In one embodiment of the present invention, the invention is focused on the synthesis and characterization of polyurethanes based on DTH as the chain extender, (see structure below):
The presence of two hydroxyl groups enables DTH to be used as chain-extender in the synthesis of polyurethane. DTH being the dipeptide moiety synthesized from L-tyrosine and its deaminated metabolite desaminotyrosine, can be effectively used for chain extension for the prepolymer made from the conventional macrodiols and the diisocyanate. Phenyl alanine based chain-extenders are the diester formed by the coupling of the carboxylic acid (of the amino acid) and the hydroxyl group of the of a low molecular weight diol (e.g., ethylene glycol). Whereas, DTH based chain extender is amide product, which makes the potentially degradable under enzymatic condition.
This invention uses the conventional two-step polyurethane synthesis process to synthesize the L-tyrosine based polyurethanes, In the first step macrodiols are reacted with diisocyanate in presence of catalyst with DMF (dimethyl formamide) as solvent at temperature 100° C. to 120° C. for 3 to 4 hours. In the second step the reaction mixture was cooled down to room temperature and the DTH chain-extender was added. The reaction was further allowed to continue for 10 to 12 hours at 70° C. to 80° C. At the end, the reaction was quenched by pouring the reaction mixture in cold concentrated solution of sodium chloride. The product was either filtered or centrifuged according to the condition of the polymer.
The macrodiols (polyols) used for the synthesis of the polyurethanes are, in one embodiment, polyethylene glycol (PEG) and poly caprolactone (PCL) based diols. Potentially non-toxic aliphatic diisocyanate hexamethylene diisocyanate (HDI) was used as the diisocyanate and DTH was the chain-extender. Two polyurethanes were synthesized as is explained in detail below using this combination as shown in the Table 3 below.
In one embodiment, the polyurethanes formed in accordance with the methods of the present invention are useful for various biomedical applications including, but not limited to, bio-scaffolding applications. Polyurethanes for biomaterial applications have been investigated for variety of applications. One criteria for such polyurethanes depends on the biocompatibility of the components used to form the polyurethane.
In one embodiment, the polyurethanes of the present invention are formed as mentioned above, and discussed in further detail below, using biocompatible polyols that include, but are not limited to, polyethylene glycol (PEG), polytetramethylene glycol (PTMG), polycaprolactone diol (PCL), or suitable combinations of two or more thereof. Several aromatic and aliphatic diisocyanates can be used in conjunction with the present invention. Such aromatic and aliphatic diisocyanates include, but are not limited to, 4,4′-diphenylmethane diisocyanate (MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), and suitable combinations of two or more thereof. Suitable chain extenders are 1,4-butanediol (BD), ethylenediamine (EA), desaminotyrosyl hexyl ester (DTH), or combinations thereof.
In one embodiment, as is mentioned above, the polyurethanes of the present invention are formed using desaminotyrosyl hexyl ester (DTH) as the chain extender. This permits the incorporation of an amino acid, or amino acid functionality, into the polyurethanes of the present invention. In another embodiment, the amino acid portion can be incorporated into the polyurethane structure as diisocyanate or the chain extender.
The synthesis of segmented polyurethanes involves two steps: (i) first the reaction of polydiol with diisocyanate in a stoichiometric ratio such that isocyanate terminated prepolymer is formed; and (ii) the reaction of the isocyanate terminated prepolymer with a low molecular weight diol or diamine compound to extend the chain.
Two different polyols are used in the present invention: polyethylene glycol (Mw 1000) (PEG) and polycaprolactone diol (Mw 1250) (PCL) because of the biocompatible characteristics of the segments formed therefrom. It should be noted that the present invention is not limited to just the compounds, or the molecular weights, given above. Instead a wide range of PEG and PCL molecular weights can be used in conjunction with the present invention to for a desired polyurethane.
In one embodiment, the diisocyanate used is aliphatic hexamethylene diisocyanate (HDI) due to its potential biocompatibility. The chain extender is desaminotyrosyl hexyl ester (DTH) is a diphenolic, dipeptide molecule based on L-tyrosine and its metabolite, desaminotyrosine (DAT).
The synthesis of polymer involves two steps: (i) synthesis of the chain extender DTH and (ii) synthesis of the polyurethane. All the chemicals and solvents were used as received, unless otherwise stated and were purchased from Sigma Aldrich. Distilled water was used for all purposes.
DTH Synthesis:
The synthesis of DTH is known to those of ordinary skill in the art and is described in various literature sources. Briefly, DTH is synthesized from hexyl ester of L-tyrosine (TH) and desaminotyrosine through carbodiimide coupling reaction. The reaction steps are summarized below and scheme of the reaction is shown below.
Step (i) The carboxylic acid group of the L-tyrosine (0.05 mole) is esterified by 1-hexanol (50 mL) in presence of thionyl chloride (0.05 mole) at 0° C. initially, followed by reaction at 80° C. for 12 hours. The reaction product obtained after cooling down the reaction to room temperature was completely precipitated in cold ethyl ether. The product was then filtered and washed with cold ether to obtain white solid, which is the chloride salt of hexyl ester of L-tyrosine.
Step (ii): The white solid was re-dissolved in distilled water and subsequently neutralized by 0.5 M sodium bicarbonate solution till the pH of the solution is slightly basic (pH—7.5). At this point solution turns turbid due to formation of tyrosine hexyl ester (TH). Tyrosine hexyl ester (TH) was extracted in ether, and the ether was evaporated to complete dryness to obtain tyrosine hexyl ester (TH) as an off-white solid.
Step (iii): Coupling of TH with DAT was mediated through hydrochloride salt of N-ethyl-N′-dimethylaminopropyl carbodiimide (EDC.HCl). Typically TH, DAT and EDC.HCl were added in equimolar proportion in 99% pure tetrahydrofuran (THF) as solvent at 0° C. After that, the reaction was allowed to continue at room temperature for 12 hours. At the end of 12 hours, the reaction mixture was poured into four times its volume of distilled water and was extracted in the organic phase by dichloromethane (DCM).
Step (iv): The organic DCM phase was washed with 0.1 N HCl solution, 0.1 N sodium carbonate solution and concentrated sodium chloride solution to remove the by products. The organic DCM phase was dried, and the solvent was evaporated under vacuum to obtain desaminotyrosyl tyrosine hexyl ester (DTH) as yellow, viscous oil.
Synthesis of Polyurethanes:
The synthesis of polyurethane is a condensation type polymerization typically involving the reaction of isocyanate (—NCO) and hydroxyl (—OH) to form the carbamate (—NHCO) linkages. The polymerization is usually a two-step process leading to the formation of segmented polyurethane: (i) reaction of polyol with diisocyanate to form isocyanate terminated prepolymer and (ii) chain extension through the reaction of prepolymer and chain extender. Two different polyurethanes were synthesized using PEG and PCL as the polyol with HDI (diisocyanate) and DTH (chain extender). The reactions were carried out in a completely dry and moisture-free environment under inert (completely dry nitrogen, N2) atmosphere. Both PEG and PCL were dried under vacuum for 48 hours at 40° C. to remove entrapped water. N,N′-Dimethyl formamide (DMF) used as solvent, was dried over calcium hydride (CaH2) followed by molecular sieve.
Diisocyanate of high (>99%) purity grade was used. The detailed protocol for the synthesis of polyurethane is summarized below:
Step (i): The polyol (PEG or PCL) was reacted with HDI at a 1 2 molar ratio in DMF as solvent and 0.1% stannous octoate catalyst to form the prepolymer. Typically, 5 mmol of polyol was added into 40 ml of DMF and 10 mmol of HDI and 2 to 3 drops of stannous octoate was added to the reaction mixture under dry and inert atmosphere with continuous stirring.
Step (ii): The temperature was increased to 110° C. and the reaction was allowed for 3 hours at this temperature. After 3 hours, the reaction cooled down to room temperature (−25° C.) with continuous stirring. The temperature of reaction was carefully maintained within the range of ±3° C.
Step (iii): DTH was added in the second step at a 1:1 molar ratio with the prepolymer. Typically, 5 mmol of DTH in 10 mL of DMF was added.
Step (iv): The temperature of reaction was then gradually increased to 80° C. and the reaction was allowed to continue for 12 hours. The temperature of reaction was controlled within the range of ±3° C. After 12 hours the reaction was quenched by pouring the reaction into cold concentrated aqueous solution of sodium chloride. At this point, solid polyurethane polymer precipitates out from the reaction mixture.
Step (v): For PEG based polyurethanes, the polymer is suspended in the form of gel in the water. The final polymer is centrifuged out and re-suspended in water and then centrifuged. This process is repeated for at least three times to remove the impurities and unreacted materials. The final polymer is then dried in vacuum at 40° C. for 48 hours. The polymer is yellowish white sticky solid. The nomenclature used for the PEG based polyurethane is PEG-HDI-DTH.
Step (iv): For PCL based polyurethanes, the polymer is suspended as solid polymer. The final polymer is filtered out and washed with water. This washing is repeated for at least three times to remove the impurities and unreacted materials. The final polymer is then dried in vacuum at 40° C. for 48 hours. The polymer is yellowish white solid. The nomenclature used for the PCL based polyurethane is PCL-HDI-DTH.
The polyurethanes synthesized were stored is desiccators for the purpose of characterization and future experiments. The structure of the two polyurethanes is shown below.
where n is an integer in the range of about 5 to about 25, m is an integer in the range of 1 to about 4, and p is an integer in the range of about 20 to about 200. In another embodiment, m is equal to 1. In still another embodiment, n is an integer in the range of about 7 to about 22, or an integer from about 10 to about 20, or even an integer from about 12 to about 17. In still another embodiment, p is an integer in the range of about 30 to about 180, or an integer in the range of about 50 to about 175, or an integer from about 75 to about 150, or even an integer from about 100 to about 125. Here, as well elsewhere in the specification and claims, individual range limits can be combined to form additional non-disclosed ranges.
In still yet another embodiment, m, n and p are selected so that the molecular weight of the above polyurethane compounds is in the range of about 4,000 Da to about 1,000,000 Da, or from about 5,000 Da to about 900,000 Da, or from about 10,000 Da to about 800,000 Da, or from about 30,000 Da to about 750,000 Da, or from about 50,000 Da to about 600,000 Da, or from about 75,000 Da to about 500,000 Da, or from about 100,000 Da to about 400,000 Da, or from about 150,000 Da to about 350,000 Da, or from about 200,000 Da to about 300,000, or even from about 225,000 Da to about 250,000 Da. Here, as well elsewhere in the specification and claims, individual range limits can be combined to form additional non-disclosed ranges. In another embodiment, m, n and p are selected so that the molecular weight of the PEG-HDI-DTH is about 98,000 Da. In another embodiment, m, n and p are selected so that the molecular weight of the PCL-HDI-DTH is about 246,000 Da.
Characterization of Polymer:
The polymerization and the polyurethanes were characterized by various techniques to determine the structure and understand the basic properties of the polymers. The preliminary characterization studies include structural, thermal and mechanical characterization.
Structural Characterizations:
The structural characterizations were done by 1H-NMR, 13C-NMR and FT-IR study. NMR was carried out in 300 MHz Varian Gemini instrument with d-dimethyl sulfoxide (δ=2.50 ppm for 1H NMR and 39.7 ppm for 13C NMR as internal reference) solvent for PEG-HDI-DTH and &chloroform (δ=7.27, ppm for 1H NMR and 77.0 ppm for 13C NMR as internal reference) for PCL-HDI-DTH. FT-IR analysis was performed with a Nicolet NEXUS 870 FT spectrometer for neat samples with 16 scans. FT-IR analysis was also used to study the progress of polymerization reaction. The molecular weights of polymers were determined by gel permeation chromatography (GPC) using tetrahydrofuran (THF) as solvent and polystyrene as internal standard. The solubility of the polymers was checked in a variety of solvents by dissolving 10 mg of solid polymer in 10 mL of the solvent at room temperature.
Thermal Characterizations:
The thermal behaviors of the polyurethanes were characterized by differential scanning calorimetry (DSC) and thermo gravimetric analysis (TGA). DSC was performed with a DSC Q100V7.0 Build 244 (Universal V3. 7A TA) instrument at a scanning rate of 10° C./min from −80° C. to 250° C. TGA was performed with a TGA Q50V5.0 Build 164 (Universal V3. 7A TA) instrument from 0° C. to 600° C. under nitrogen atmosphere at a rate of 20° C./min. An average of 10 mg of solid sample was used for both the experiments.
Mechanical Characterizations:
The tensile properties of the polyurethanes films were measured by Instron Tensile Testing Machine with a load cell of 100 N and cross-head speed of 100 mm/min at room temperature. The films were cast from 10% wt solution of polymers (DMF for PEG-HDI-DTH and chloroform for PCL-HDI-DTH) and solvent was allowed to evaporate at room temperature and then subsequently dried in vacuum oven at 50° C. for 48 hours to remove the residual solvent. The sample dimension was 20 mm×6 mm×−0.3 mm with a free length of 10 mm. The average of five measured values was taken for each sample.
Polymerization Reaction:
Table 4 summarizes the composition of the two polymers with the relative contribution of hard and soft segment. The yield for the synthesis of DTH was about 85% and for the polyurethanes was about 70 to 80%. The results were reproducible within a range of ±5% with reasonable purity of the polyurethanes.
NMR Characterization:
The 1H NMR (along with the peak assignments) of PEG-HDI-DTH and PCL-HDI-DTH is shown in
PCL-HDI-DTH: δ 0.8 (CH3— in hexyl group, DTH), 1.2 to 1.7 (CH2 in DTH, HDI and PCL), 2.3 (—CO—CH2— in PCL), 2.8 (—CH2—CH2—CO— in DTH), 2.9 (—NH—CH2— in HDI and —C6H4—CH2— in DTH), 3.1 (—C6H4—CH2—CH in DTH), 4.0 (—CO—O—CH2—CH2— in DTH and PCL), 4.8 (—NH—CH—(CO)—CH2— in DTH), 6.7 and 6.9 (two —C6H4— in DTW).
The 13C NMR (along with the peak assignments) of PEG-HDI-DTH and PCL-HDI-DTH is shown in
PCL-HDI-DTH: δ 14.2 (CH3— in hexyl group, DTH), 22.7 to 28.6 (CH2 in hexyl chain in DTH, HDI, PCL), 29.9 to 31.5 (CH2 in hexyl chain in DTH, HDI), 34.1 (—CH2—CH2—CO in DTH), 34.3 (—CH2—CH2—CO—O in PCL), 40.5 (—C6H4—CH2—CH in DTH), 53.5 (—NH—CH—(CO)—CH2— in DTH), 64.3 (—CO—O—CH2—CH2— in PCL), 129.5 and 130.4 (two —C6H4— in DTH), 156.0 (—NH—CO—O— in urethane carbonyl) and 172.0 (ester and amide carbonyls in DTH), 173.7 (ester carbonyls in PCL).
The peak assignment from 1H and 13C NMR show that all the three components are present in the polymer chains. However due to the presence of similar chemical environments for certain protons and carbons, there is considerable overlap of the peaks which makes the assignment a difficult task. In general, for both the PEG- and PCL-based polyurethanes the presence of the characteristic peaks indicate that the polymers are composed of the corresponding soft segments along with HDI and DTH. Most important is the presence of urethane link indicated by the 2.9 ppm in 1H NMR and 156 ppm in 13C NMR for both in PEG- and PCL-based polyurethanes. This clearly shows that urethane linkages are formed by the condensation polymerization. However some unassigned peaks in the spectra correspond to materials formed by possible side reactions and from of unreacted materials/solvent. But the intensity of such peaks are considerably lower than the assigned peaks which indicates that polymers are of reasonable purity.
FT-IR Characterizations:
The FT-IR spectra of the polyurethanes are shown in
The FT-IR of the starting materials, intermediate prepolymer and the final polymer is shown together in
Table 5 summarizes the molecular weight of the polymers which shows that both the polyurethanes have significantly high molecular weight. Compared to the molecular weight of PEG and PCL as starting material, the molecular weight of the final polymers indicates the formation of polyurethanes. The low poly-dispersity indices of the polyurethanes indicate that the distribution of molecular weight is not broad and the polymerization is controlled. However, PEG based polyurethanes are lower in molecular weight compared to PCL based polyurethanes. While not wishing to be bound to any one theory, this is probably due to presence of residual water in precursor PEG which inhibits high molecular weight of polymer by reacting away the diisocyanate. Considering different factors that contribute to the molecular weight of polymers in solution polymerization, these results were reproducible within range of ±10%.
Solubility of the Polyurethanes:
Table 6 shows the solubility features of the polyurethanes in the common solvents.
The solubility of the polymers shows that the polyurethanes are soluble in polar aprotic solvents and insoluble in water and protic solvents. The polyurethanes are also insoluble in acetone, ethyl acetate which is polar and aprotic, indicating that the different phases of the polyurethanes contribute differently towards solubility. But in general, the solubility features indicate that the polyurethanes are soluble for practical purposes.
Thermal Characterizations:
The DSC thermograms of the polyurethanes are shown in
The DSC thermograms of the polyurethanes show distinct glass transition (Tg) at −40° C. for PEG-HDI-DTH and at −35° C. for PCL-HDI-DTH which correspond to the soft segment glass transition temperature. The shift from the Tg's of the pure homopolymer Tg's (−67° C. for PEG and −62° C. for PCL) indicates some degree of phase mixing between the soft and hard segment of the polyurethanes. For PEG-HDI-DTH, three additional endotherms were observed: at 0, 50 and 162° C. Similar endotherms are also observed for PCL-HDI-DTH at 5, 52 and 173° C. with an additional one at 31° C. The absence of hard segment Tg indicates that hard segments are relatively crystalline domains due to presence of aromatic ring structure in the back bone of polymer. It has been observed a hard segment Tg that is probably due to amorphous hard segment with aromatic group as pendant groups from the backbone of the polymer.
Moreover, absence of melting endotherms for the phenyl alanine based polyurethanes indicates that the hard segment is largely amorphous. The endotherms at 162° C. represent the melting of the microcrystalline hard segment domain while the other transitions at 0 and 50° C. represents the dissociation of short range and long range order of the hard segment domain. Short range order of polyurethane actually represents the interaction between the soft segment and hard segment that actually contributes to the phase mixing behavior of the polyurethane. Long range order represents ‘unspecified’ interactions within the hard segment domain. Absence of soft segment melting endotherm for PEG-HDI-DTH indicates the amorphousness of the soft segment. The crystallinity of PEG is reduced due to the presence of hard segment at the PEG chain ends and due to partial dispersion of the hard segment within the soft segment of the polyurethane. The low molecular weight of PEG and high hard segment content in PEG-HDI-DTH favors this feature. Similar observations for PTMO based polyurethanes and phenyl alanine based polyurethanes are made. The similar endotherms for PCL-HDI-DTH at 173° C. represent the melting of the microcrystalline hard segment domain while the other transitions at 0 and 52° C. represents the dissociation of short range and long range order of the hard segment domain respectively.
The additional endotherm at 31° C. is probably due to the melting of soft segment. PCL being relatively more crystalline shows melting due to chain mobility at this temperature. The crystallinity of PCL soft segment is less affected in spite of phase mixing due to the dipolar interaction of ester bonds and relatively lower hard segment content. The phase mixing phenomenon is present in both the polyurethanes but PCL based polyurethane exhibits comparatively lesser degree of mixing than PEG-based polyurethane. The crystalline PCL soft segment is more cohesive in nature which prevents the mixing of hard and soft segment at the molecular level whereas relatively amorphous and non-polar PEG soft segment provides more integration in between the different segments. These characteristic features of the polyurethanes indicate that two phase morphology of the polyurethanes are present with variable degree of phase mixing/segregation behavior. The relative crystallinity of the polymers is mainly contributed by the H-bonded hard segment. The DSC analysis of the polyurethanes provides significant information about phase morphology of the polyurethanes.
The thermogravimetric analysis (TGA) analysis of the polyurethanes is shown in
The melting of the polymers is at relatively lower temperature compared to pure poly-tyrosine indicates its applicability in the processing of the material for practical purposes of scaffolding in tissue engineering applications. The high degradation temperature indicates that the range of temperature within which the polymers are processible is sufficiently large.
Mechanical Characterizations:
The typical stress-strain curve of the polyurethanes is shown in
The mechanical properties of polyurethanes show that PEG based polyurethane is lower in mechanical strength compared to PCL based polyurethane. The mechanical properties of the polyurethanes are mainly controlled by the dominant soft segments. The lower tensile strength, modulus of elasticity and elongation (at break) of PEG-HDI-DTH is largely due to amorphous and flexible PEG soft segment compared to relatively more crystalline PCL. The contribution of hard segment is relatively less due to phase mixing of the hard segment with the soft segment. Thus, the mechanical properties of the polyurethanes are more controlled by the soft segment morphology. The difference in the mechanical properties of the polyurethanes can be directly correlated to structure and morphology of the polyurethanes. Polyurethanes with higher degree of phase separation exhibits better tensile properties than the phase mixed polyurethanes. This is probably due to disordering of hard segment domains. As indicated by DSC analysis, crystalline PCL soft segment inhibits phase mixing and therefore leads to more phase segregated morphology leading to higher tensile properties. In addition to this, the effect of molecular weight is directly related to the tensile property. PCL based polyurethane have significantly higher molecular weight which improves the tensile properties compared to the PEG based polyurethane. Moreover, the high hydrophilicity of PEG often leads to lower mechanical property of the polymer.
Preliminary physical and chemical characterization indicates that polyurethanes can be synthesized using DTH as the chain extender. 1H and 13C NMR shows the presence of aromatic moieties which conclusively proves the inclusion of DTH as the chain extender. Moreover, the IR characterization shows appearance of urethane, and amide groups (1650 to 1700 cm−1) for the polymer. The GPC analysis primarily concludes the polymerization process with sufficiently high molecular weight and relatively narrow molecular weight distribution. The solubility studies show that these polymers are partially to completely soluble in most the solvents.
The thermal characterization studies indicates both the polymers have melting temperature around 150° C. (from DSC analysis), whereas the onset of the decomposition is around 300° C. (from TGA analysis). These results indicate the wide thermal range for the processing of the material.
The hydrolytic degradation of the polymer at physiological pH 7.4 and body temperature 37° C. shows that PEG based polyurethanes are degradable under these conditions whereas PCL based polyurethanes are potentially less degradable under similar conditions. These results were further supported by the low water uptake of PCL based polyurethanes (approximately 5%) compared to their PEG (approximately 70%) based counterpart.
Conclusions:
The preliminary results of the L-tyrosine based polyurethanes indicate that these polymers can be synthesized easily by two-step methods. The characterization results indicate that these materials are suitable for biomaterial applications. Further and elaborate characterizations, including mechanical and biological properties of these polymers are currently under investigation for tissue engineering applications. This invention shows that L-tyrosine based DTH can be used as chain extender for the synthesis of polyurethanes. These polymers have the potential for biomaterial applications.
Based on the present invention, L-tyrosine-based phosphate polymers can be synthesized that degrade over shorter period of times, for example, in less than 20 days, less than 15 days, or even less than 7 to 10 days. On the other hand, the present invention also makes it possible to synthesize L-tyrosine-based urethane polymers that degrade over a period of several months to one year. In another embodiment, the present invention makes it possible to form copolymers of L-tyrosine-based phosphate polymers and L-tyrosine-based urethane polymers thereby permitting one to further control the degradation rate thereof.
In another embodiment, the present invention relates to a blended L-tyrosine polyphosphate polymer with a L-tyrosine polyurethane polymer. In this embodiment, depending upon the composition of each component in the blend it is possible to achieve a wide variety of degradation times. While not wishing to be bound to any one embodiment, as a general rule the L-tyrosine polyphosphate polymers of the present invention degrade in less time (e.g., usually less than about 2 weeks, less than about 7 days) that the L-tyrosine polyurethane polymers disclosed herein (degradation time of about 1 months to several months or longer). Thus, based upon the percentage of each in a polymer blend one could achieve a wide range of desired degradation times for a wide variety of biomedical applications.
In another embodiment, the present invention relates to homopolymers, co-polymers, or blended polymers mixtures of various L-tyrosine polyphosphate polymers as disclosed herein. In still another embodiment, the present invention relates to homopolymers, co-polymers, or blended polymers mixtures of various L-tyrosine polyurethane polymers as disclosed herein.
Although the invention has been described in detail with particular reference to certain embodiments detailed herein, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and the present invention is intended to cover in the appended claims all such modifications and equivalents.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2007/024075 | 11/16/2007 | WO | 00 | 3/29/2011 |
Number | Date | Country | |
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60859436 | Nov 2006 | US | |
60959731 | Jul 2007 | US |