Clyde M. Ofner

The determination of ϵ-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometrie method using trinitrobenzenesulfonic acid

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.

Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices

AbstractPurpose. To determine the extent of amino group crosslinking in gelatin matrices by chemical assay, and to compare these results to crosslinking evaluations from swelling measurements. Methods. Matrices crosslinked with a water soluble carbodiimide (EDC/G), glutaraldehyde (GTA/G), as well as a GTA crosslinked matrix prepared from gelatin modified to contain 230% greater crosslinking sites (GTA/Mod) were evaluated. Crosslinking extent, Xc, was determined by a UV assay of uncrosslinked amino groups before and after crosslinking, and was used to obtain crosslinking densities. Equilibrium swelling ratios, Qm at 37°C in isotonic pH 7.4 were used to calculate crosslinking degree from the Flory equation for swelling of ionic polymers for comparison to the chemically determined crosslinking densities. Results. Of the original 33 × 10−5 moles ε-amino groups/g gelatin, 91 to 95% were crosslinked in EDC/G and GTA/G. GTA/Mod lost 95% of the original 108 × 10−5 moles amino groups/g gelatin. Crosslinking densities were 4.1 × 10−4 and 4.2 × 10−4 moles/mL for EDC/G and GTA/G, respectively. The value for GTA/Mod increased to 14.2 × 10−4 moles/mL. Values of Qm followed the same trend. The Flory crosslinking degrees for both gelatin matrices were 12 × 10−4 and 13 × 10−4 moles/mL, respectively. The value for the more extensively crosslinked GTA/Mod was 280 × 10−4moles/mL. Conclusions. The swelling and chemical evaluations of crosslinking are in general agreement for matrices with the lower of two crosslinking levels. The chemical determination appears suitable for evaluating amino group crosslinking in gelatin and it may be suitable for other proteinaceous materials.

Crosslinking Studies in Gelatin Capsules Treated with Formaldehyde and in Capsules Exposed to Elevated Temperature and Humidity

Incomplete in vitro capsule shell dissolution and subsequent drug release problems have recently received attention. A modified USP dissolution method was used to follow capsule shell dissolution, and a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay was used to follow loss of epsilon-amino groups to study this shell dissolution problem postulated to be due to gelatin crosslinking. The dissolution problems were simulated using hard gelatin capsule (HGC) shells previously treated with formaldehyde to crosslink the gelatin. These methods were also used to study the effect of uncrosslinked HGC stored under stressed conditions (37 degrees C and 81% RH) with or without the presence of soft gelatin capsule shells (SGC). A 120 ppm formaldehyde treatment reduced gelatin shell dissolution to 8% within 45 min in water at 37 degrees C. A 200 ppm treatment reduced gelatin epsilon-amino groups to 83% of the original uncrosslinked value. The results also support earlier reports of non-amino group crosslinking by formaldehyde in gelatin. Under stressed conditions, HGC stored alone showed little change over 21 weeks. However, by 12 to 14 weeks, the HGC exposed to SGC showed a 23% decrease in shell dissolution and an 8% decrease in the number of epsilon-amino groups. These effects on the stressed HGC are ascribed to a volatile agent from SGC shells, most likely formaldehyde, that crosslinked nearby HGC shells. This report also includes a summary of the literature on agents that reduce gelatin and capsule shell dissolution and the possible mechanisms of this not-so-simple problem.

Characterization and in vitro release of methotrexate from gelatin/methotrexate conjugates formed using different preparation variables

The purpose of this study was to evaluate effects of preparation variables on the composition of gelatin-methotrexate conjugates, and to evaluate their in vitro stability. Conjugation variables of pH, amount of conjugating agent 1-ethyl-3-(diaminopropyl)carbodiimide HCl (EDC), and methotrexate (MTX), with unfractionated gelatin were examined. Conjugate composition was determined spectrophotometrically. The molar ratios of MTX to gelatin in the conjugates ranged from 5.9 to 64.9. Molar ratios increased with molecular weight (MW) of gelatin in the conjugate, but the weight ratio was constant. This common conjugating procedure, however, produces by-product crosslinking and produces a mix of covalent MTX binding to carboxyl and amino groups of the gelatin. For release studies, gelatin was fractionated by size exclusion spectra (SEC) into MW of 21, 91, and 195 kDa prior to conjugation. MTX release from conjugates in dialysis cassettes at 25, 37, and 50 degrees C, in isotonic pH 7.4, buffer over 72 h was assayed by high performance liquid chromatography (HPLC). There was no effect of gelatin MW on MTX release. MTX release was approximately linear and attained 2.3, 7.2, and 13% by 72 h at 25, 37, and 50 degrees C, respectively, for the 91 kDa conjugates. First-order release rate constants were 0.23 x 10(-3), 0.95 x 10(-3), and 1.8 x 10(-3) x h(-1), respectively. The calculated activation energy for MTX release was 15.8 kcal/mol. Rate constants and the activation energy for MTX release are consistent with hydrolysis of a peptide bond. Non-degraded MTX species were found in the release medium at amounts similar to free MTX and were attributed to MTX polymers and MTX/gelatin fragments < 10 kDa.

Microencapsulation of chlorpheniramine maleate-resin particles with crosslinked chitosan for sustained release.

Formulation and preparation parameters of drug/ion-exchange particles microencapsulated in cross-linked chitosan were evaluated for controlled release of the water-soluble drug chlorpheniramine maleate (CPM) in a suspension. An emulsion solvent evaporation method was used to produce CPM-resinates embedded in glutaraldehyde (GTA) crosslinked chitosan microspheres (MCSs). Crosslinking extent in the chitosan was monitored by swelling measurements. Controlled release was evaluated by dissolution tests in simulated gastric fluid without enzyme (SGF) and in simulated intestinal fluid without enzyme (SIF). CPM-resinates contained 62% (w/w) of drug. MCSs were spherical, ranging from 82 to 420 microns in diameter, and contained multiple resinates. The sizes of MCSs prepared with safflower oil and Span 80 were controlled by surfactant concentration, stirring speed, and duration of stirring. Maximum crosslinking was produced with 240 mg GTA per 250 mg of chitosan. Maximum drug release from free CPM-resinates was about 60% by 1 hr in SGF, and was about 100% by 3 hr in SIF. CPM release was slower from MCSs crosslinked with 120 mg of GTA compared to 5 mg GTA in both media. By 8.3 hr, the more crosslinked MCSs released about 30% CPM in SGF, and about 60% in SIF. Because of the apparent ceiling on release in SGF, the final experiments were conducted in SIF. Increasing the weight ratio of the chitosan coating to CPM-resinate ratio from 1:1 to 4:1 moderately decreased release profiles carried out to 33 hr. Increasing MCS diameters from 82 to 163 microns moderately decreased release profiles. Microencapsulation of CPM-resinates with crosslinked chitosan demonstrated controlled release of CPM in SGF and SIF without enzymes. The retardation effect increased when the crosslinking extent and chitosan to resin ratio increased.

Carbodiimide induced cross-linking, ligand addition, and degradation in gelatin.

The water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide (EDC) is widely used in protein chemistry. We used EDC-induced gelatin cross-linking as a model for amide bond formation to resolve reaction ambiguities with common variables of buffers, gelatin concentration, and pH. Percentage changes in SEC high molecular weight peak areas were used to follow the reactions. Differences in reaction rate and extent were observed with four commonly used buffers, while differences in extent were observed for commonly used concentrations and pH. We also investigated an anhydride mechanism for aqueous EDC-induced amide bond formation that has received little attention since its proposal in 1995. Gelatin carboxyl groups had a synergistic role during the addition of hydrazine to corroborate the anhydride formation between carboxyl groups. EDC-induced degradation of gelatin was investigated using percentage changes in SEC low molecular weight peak areas. The degradation occurred in excess EDC at neutral to alkaline pH and was enhanced substantially when reacting amino groups were not available. A mechanism of EDC-induced gelatin degradation is proposed and designated the extended Khorana mechanism. This EDC side reaction has the potential to occur in peptides and proteins under similar conditions.

Characterization and in vitro methotrexate release from methotrexate/gelatin conjugates of opposite conjugate bond polarity.

AbstractPurpose. Our laboratory has previously prepared gelatin/methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking. Methods. Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37°C for 7 days. Results. The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76×10−3 and 1.0×10−3 hr−1, respectively. Alternate MTX species (≤ 10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers. Conclusions. Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.

Charge-transfer complexes of iodine and nonionic surfactants: Interpretation and use in the Winkler method☆

Formation constants (Kc) and molar absorption coefficients (epsilon c) of complexes of iodine and various nonionic surfactants were determined, providing a basis for selection of a surfactant for use in a spectrophotometric modification of the Winkler method. The method of calculation of Kc and epsilon c was extended to include absorption by triiodide at the wavelength of maximum absorbance of the complex. Because the molar absorption coefficients of polyoxyethylene 10 oleyl ether (oleth 10) and polyoxyethylene 23 oleyl ether were significantly greater than those of other surfactants, they are superior candidates for use in the Winkler method. Formation constants could not be correlated with molecular characteristics of the surfactants such as alkyl chain or polyoxyethylene chain length, nor with physical characteristics of iodine-surfactant solutions such as reduction of iodine loss due to volatilization.