Masawo Kakemi

Controlled release of plasmid DNA from cationized gelatin hydrogels based on hydrogel degradation

This paper shows achievement of the in vivo controlled release of a plasmid DNA from a biodegradable hydrogel and the consequent regulation of gene expression period. Cationization of gelatin was preformed through introduction of ethylenediamine and the gelatin prepared was crosslinked by various concentrations of glutaraldehyde to obtain cationized gelatin (CG) hydrogels as the carrier of plasmid DNA. In vivo release of plasmid DNA from the CG hydrogels was compared with the in vivo degradation of hydrogels. When CG hydrogels incorporating 125I-labeled plasmid DNA were implanted into the femoral muscle of mice, the plasmid DNA radioactivity remaining decreased with time and the retention period prolonged with a decrease in the water content of hydrogels used. The higher the water content of 125I-labeled CG hydrogels, the faster the hydrogel radioactivity remaining decreased with time. The time profile of plasmid DNA remaining in the hydrogels was in good accordance with that of hydrogel radioactivity, irrespective of the water content. Intramuscular implantation of plasmid DNA-incorporated CG hydrogels enhanced significantly expression of the plasmid DNA around the implanted site. The retention period of gene expression became longer as the hydrogel water content decreased. Fluorescent microscopic study revealed that the plasmid DNA-CG complex was detected around the hydrogel implanted even after 7-day implantation in marked contrast to the injection of plasmid DNA solution. It was concluded that in our hydrogel system, active plasmid DNA was released accompanied with the in vivo degradation of hydrogel, resulting in extended gene expression. The time profile of plasmid DNA release and the consequent gene expression was controllable by changing the water content of hydrogels.

Oral delivery of insulin by using surface coating liposomes: Improvement of stability of insulin in GI tract

The potency of surface coating liposomes with some materials was investigated for oral delivery of peptide drugs. In vitro release of insulin, a model peptide, from liposomes in the bile salts solution was markedly reduced by coating the surface with the sugar chain portion of mucin (Mucin-Lip) or polyethyleneglycol (PEG-Lip). Encapsulation of insulin into Mucin-Lip and PEG-Lip completely suppressed the degradation of insulin in the intestinal fluid, whereas uncoated liposomes suppressed it only partially. These results demonstrated that surface coating liposomes with PEG or mucin gained resistance against digestion by bile salts and increased the stability in the GI tract. When insulin was orally administered to rats as either a solution or non-charged liposome ((N)-Lip), no hypoglycemic effect was observed. Administration of insulin encapsulated in positively charged liposome ((+)-Lip) caused the rapid decrease in the plasma glucose level which recovered to the control level within 3 h. In contrast, PEG-Lip and Mucin-Lip caused a gradual decrease in the glucose level after administration. The hypoglycemic effect by PEG-Lip lasted for much longer duration than that of uncoated liposomes. The slow release of insulin from the surface coating liposomes achieved the longer duration of oral hypoglycemic activity. Consequently, the surface coating should be the potential way to add desirable functions to the liposome for oral drug delivery.

In vivo release and gene expression of plasmid DNA by hydrogels of gelatin with different cationization extents

The objective of this paper is to investigate the in vivo release and gene expression of lacZ plasmid DNA (pSV-lacZ) by the hydrogels of cationized gelatin. Gelatin with different cationization extents was prepared by changing the amount of ethylenediamine added to aminize the carboxyl groups of gelatin with a water-soluble carbodiimide. The cationized gelatin prepared was crosslinked by various concentrations of glutaraldehyde (GA) to obtain cationized gelatin hydrogels with different cationization extents as the release carrier of plasmid DNA. When the cationized gelatin hydrogels incorporating 125I-labeled pSV-lacZ were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period was prolonged with an increase in the concentration of GA used for hydrogel preparation. In vivo experiments with 125I-labeled cationized gelatin hydrogels revealed that the higher the GA concentration, the longer the in vivo retention period of radioactivity remaining for every cationized gelatin hydrogel. Only for the hydrogels prepared from gelatin with the aminized percentages of 29.7, 41.6, and 47.8 mol.%, the time profile of pSV-lacZ retention correlated well with that of hydrogel retention. The gene expression by the cationized gelatin hydrogels incorporating pSV-lacZ depended on the aminized percentage of gelatin and was significant at the percentage of 41.6 mol.% or higher. It is possible that the pSV-lacZ was complexed with the degraded fragments of cationized gelatin and released with a positive charge, resulting in enhanced gene expression. We conclude that gelatin with a cationization extent of at least 41.6 mol.% is needed for the enhanced in vivo gene expression of plasmid DNA by the hydrogel release system.

Characterization of organogel as a novel oral controlled release formulation for lipophilic compounds

A low molecular mass gelator can form soft solids in a variety of organic liquids and vegetable oils. These soft solids are generally called organogels. In this study, we prepared organogel using 12-hydroxystearic acid (12-HSA) as a gelator for soybean oil and investigated its characteristics as a controlled release formulation for lipophilic compounds. The release rate of ibuprofen, a model lipophilic compound, from organogel decreased with the increase of 12-HSA concentration in the formulation; however, the difference in the concentration of 12-HSA in the formulation did not affect the diffusivity of ibuprofen in the organogel. The erosion constant of organogel in the intestinal tract was examined by using simulated gastric fluid and intestinal fluid. Regardless of 12-HSA concentration in the formulation, organogel is very stable in the simulated gastric fluid. On the other hand, the erosion constant of organogel in the simulated intestinal fluid increased with the decreasing concentration of 12-HSA. Therefore, it is speculated that the difference in the release rate of ibuprofen among organogels with various concentrations of 12-HSA was mainly caused by the difference in the erosion rate. To characterize the organogel effect in vivo, ibuprofen was orally administered to rats in an aqueous suspension or organogel. Ibuprofen concentration in plasma rapidly increased after administration with an aqueous suspension, whereas organogel suppressed the rapid absorption. In conclusion, organogel is clearly useful as an oral controlled release formulation for lipophilic compounds.

Evaluation of gelatin microspheres for nasal and intramuscular administrations of salmon calcitonin

The suitability of gelatin microspheres for nasal and intramuscular delivery of salmon calcitonin (sCT) was examined. Negatively and positively charged gelatin microspheres were prepared using acidic gelatin [isoelectric point (IEP) value of 5.0] and basic gelatin (IEP=9.0), respectively. The average diameters of positively charged gelatin microspheres in their dried state were 3.4, 11.2, 22.5 and 71.5 microm, while that of negatively charged gelatin microspheres was 10.9 microm. Both types of gelatin microspheres were capable of adhering to the nasal mucosa. The mucoadhesion of positively charged gelatin microspheres was significantly higher than that of their negatively charged counterparts. The absorption of sCT after intranasal and intramuscular administration was evaluated by calculating the area above the hypocalcemic-time curve (AAC) in rats. The AAC values after nasal administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that in pH 7.0 PBS. Therefore, the nasal absorption of sCT was enhanced by both types of gelatin microspheres. The hypocalcemic effect after administration of sCT in positively charged gelatin microspheres of 11.2 microm was significantly greater than that of negatively charged gelatin microspheres of the same size. On the other hand, AAC values were not affected by their particle sizes. The AAC values after the intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly increased compared to that in PBS. Furthermore, the time-courses of the plasma calcium levels differed between positively and negatively charged gelatin microspheres. The hypocalcemic effect of the negatively charged gelatin microspheres tended to appear more slowly and last longer compared to that of positively charged gelatin microspheres. The hypocalcemic effects after intramuscular administration of sCT in gelatin microspheres were not affected by their particle sizes as well as those after intranasal administration. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT.

Surfactant-Free Preparation of Biodegradable Hydrogel Microspheres for Protein Release

The preparation of biodegradable hydrogel microspheres in the absence of surfactants was carried out by a two-step procedure which involved the formation of non-crosslinked microspheres from gelatin based on its inherent gelation nature at low temperatures and the subsequent glutaraldehyde (GA) crosslinking. The size of the microspheres was controlled in the range of 3 to 100 μm by changing the concentration of gelatin or GA, the emulsification method, and the crosslinking time. Neutral aqueous solutions of proteins with different isoelectric points (IEPs) and molecular weights (Mws) were infused into freeze-dried hydrogel microspheres to produce protein-incorporated gelatin microspheres. In vitro protein release from the microspheres depended on the proteins IEP but not on the Mw. The incorporated basic proteins with IEPs > 7.0 were released initially from the acidic gelatin microspheres, followed by no substantial release, whereas a larger initial release of the incorporated acidic proteins with IEPs < 7.0 was observed. The basic gelatin microspheres exhibited an opposite relationship between proteins IEP and protein release. Noncharged dextran rapidly diffused out of acidic gelatin microspheres, irrespective of the Mw. These findings indicate that an ionic interaction with gelatin constituted hydrogel microspheres prevented oppositely charged protein from being released from gelatin under in vitro non-degradation conditions.

Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity

To examine the systemic and mucosal immunity towards a liposomal antigen in an oral vaccine, we prepared ovalbumin (OVA)-encapsulating polyethyleneglycol (PEG)-modified liposomes and unmodified ones, and orally administered two different concentrations of them to mice. Unmodified liposomes tended to induce a stronger systemic immune response than the PEG-modified ones especially at the higher concentration of liposomes. Whereas at the lower liposome concentration the mucosal immune response was stronger for the PEG-modified liposomes than for the unmodified ones but nearly the same at the higher concentration. The relative amount of immunoglobulin G (IgG) against OVA in the plasma was 1.7-fold higher for a 12.5 micro mol phospholipid dose of PEG-liposomes encapsulating OVA than for a 5.0 micro mol one encapsulating the same amount of OVA. On the contrary, the relative amount of IgA in the intestinal wash was 2.6-fold higher for the 5.0 micro mol phospholipid dose than for the 12.5 micro mol one. These results indicate that OVA encapsulated in a small number of liposomes, especially the PEG-modified ones, is favorable for inducing a mucosal immune response and that the same amount of OVA in a large number of liposomes tends to improve the systemic immune response. A possible explanation for this tendency is the differential release rate of OVA from the liposomes at the intestinal mucosa. Our present study suggests that the dose of liposomes containing antigen is an important factor for controlling the response of systemic and mucosal immune systems.

Effects of proteolytic enzyme inhibitors on nasal absorption of salmon calcitonin in rats

Abstract Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal absorption of salmon calcitonin (SCT) in rats. Bestatin, diprotinin, leupeptin, aprotinin, soybean trypsin inhibitor and camostat mesilate were used as enzyme inhibitors. The nasal absorption of SCT was evaluated by measuring its hypocalcemic effects. The peptidase activities of rat nasal mucosal tissue were high and found to be in the following order: leucine aminopeptidase (2.72 nmol/min per mg protein) > dipeptidyl aminopeptidase (1.84 nmol/min per mg protein) > cathepsin (650 pmol/min per mg protein) > trypsin.(4.61 pmol/min per mg protein). Nasal administration of SCT (10 IU/kg, pH 7.0) showed low pharmacological availability (3.2%). Coadministration with bestatin (aminopeptidase inhibitor, 0.0 1– 1 mM) or diprotinin A (dipeptidyl peptidase inhibitor, 0.1-1 mM) did not change the hypocalcemic effects. Coadministration with aprotinin (trypsin inhibitor, 10 3 -10 4 KIU/ml), camostat mesilate (aminopeptidase and trypsin inhibitor, 0.1 – 10 mM) or leupeptin (trypsin and cathepsin B inhibitor, 0.1 – 1 mM) enhanced the hypocalcemic effects and, thus, the nasal absorption of SCT. The hypocalcemic effects of SCT at various pH values (pH 4.0, 7.0 and 8.0) with or without aprotinin were the highest at pH 4.0. The pharmacological availabilities after nasal administration of SCT (10 IU/kg) at pH 4.0 and 7.0 were increased from 5.4 to 7.5% and from 3.2 to 6.9% by aprotinin (10 4 KIU/ml), respectively. Therefore, inhibitors which have a trypsin inhibitory activity are useful for enhancing nasal absorption of SCT.

Gelatin microspheres as a pulmonary delivery system: Evaluation of salmon calcitonin absorption

The use of negatively and positively charged gelatin microspheres for pulmonary delivery of salmon calcitonin was examined in rats. The microspheres were prepared using acidic gelatin (isoelectric point (IEP):, 5.0) and basic gelatin (IEP, 9.0) for the negatively and positively charged microspheres, respectively. The average diameters of positively charged gelatin microspheres in the dry state were 3.4, 11.2, 22.5 and 71.5 μm, and that of negatively charged gelatin microspheres was 10.9 μm. Neither positively nor negatively charged gelatin microspheres underwent any degradation in pH 7.0 PBS and there was less than 8% degradation in bronchoalveolar lavage fluid (BALF) after 8 h. In in‐vitro release studies in pH 7.0 PBS, salmon calcitonin was rapidly released from positively charged gelatin microspheres within 2 h, and its cumulative release was approximately 85%. In addition, the release profiles were not influenced by particle sizes. The release rates of salmon calcitonin from negatively charged gelatin microspheres were lower than that from positively charged gelatin microspheres. The cumulative release was approximately 40% after 2 h, but there was no evidence of any sustained release.

Usefulness of microspheres composed of gelatin with various cross-linking density

The release rate of insulin, as a model peptide, from gelatin microspheres (GM) prepared with gelatin having various cross-linking densities in vitro was examined. The release of insulin from GM showed the burst effect, followed by a slow release phase regardless of the cross-linking density of gelatin. The total amount of insulin released in 2 weeks decreased with increasing cross-linking density of gelatin. The release rate of insulin within 6 h was well correlated with the cross-linking density of gelatin. The remaining amounts of both insulin and GM after injection of insulin incorporated in GM to mice femoral muscle tissue were also examined in vivo. Both insulin and GM rapidly disappeared from the injection site within 1 day, and thereafter slowly disappeared over 14 days. The time courses of the remaining amounts were fairly similar to each other. Furthermore, the remaining amount of insulin 1 day after administration was well correlated with the cross-linking density of gelatin. These data suggest that insulin was released from GM with the degradation of GM in mice muscular tissue and that the release rate of insulin can be controlled by modifying the cross-linking density of gelatin. In conclusion, the control of the release rate of insulin from GM can be achieved under both in vitro and in vivo conditions by gelatin through the alteration of cross-linking conditions.