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.

Preparation, drug release, and cell growth inhibition of a gelatin: doxorubicin conjugate.

PURPOSE: To demonstrate the feasibility of a novel macromolecular delivery system for doxorubicin (DOX) which combines pH dependent DOX release with a high molecular weight and biodegradable gelatin carrier. METHODS: DOX was conjugated to gelatin using an acid labile hydrazone bond and a glycylglycine linker. The gelatin-doxorubicin conjugate (G-DOX) was evaluated for hydrazide and DOX content by spectrophotometry, molecular weight by HPLC-SEC, in vitro DOX release at various pH, and cell growth inhibition using EL4 mouse lymphoma and PC3 human prostate cells. RESULTS: G-DOX hydrazide and DOX content was 47% and 5-7%, respectively of theoretical gelatin carboxylic acid sites. During preparation of G-DOX, the molecular weight decreased to 22 kDa. DOX release was 48% in pH 4.8 phosphate buffer, 22% at pH 6.5, but 10% at pH 7.4. The G-DOX IC50 values in EL4 and PC3 cells were 0.26 µM and 0.77 µM, respectively; the latter value 3 times greater than that of free DOX. CONCLUSIONS: A 22 kDa macromolecular DOX conjugate containing 3.4-5.0% w/w DOX has been prepared. The pH dependent drug release in combination with a biodegradable gelatin carrier offer potential therapeutic advantages of enhanced tumor cell localization and reduced systemic toxicities of the drug.

Adsorption and degradation of doxorubicin from aqueous solution in polypropylene containers.

The purpose of this study was to examine doxorubicin adsorption in polypropylene containers as a function of pH and drug concentration based on anecdotal evidence of such adsorption. Doxorubicin loss was first examined in high-performance liquid chromatography (HPLC) glass inserts by UV absorbance to determine appropriate pH and time durations for subsequent analysis. Doxorubicin loss was then investigated in polypropylene microcentrifuge tubes at different pH values and starting drug concentration at 37°C over 48 h using HPLC with fluorescent detection. Doxorubicin concentrations was essentially constant in HPLC glass inserts at pH 4.8 up to 12 h but declined 5% at pH 7.4 by 3 h. The percent doxorubicin adsorption was calculated in polypropylene microcentrifuge tubes from extrapolations to zero time and was the least at pH 4.8, but increased with pH values 6.5 and 7.4, and decreased with drug concentration to reach a maximum adsorption of 45% in 2.0 µg/mL at pH 7.4 and 37°C. Degradation rate constants, ranging from 0.0021 to 0.019 h(-1), also increased with pH in these studies. Determinations of low amounts of doxorubicin in polypropylene containers at pH 7.4 may be underestimated if adsorption and degradation issues are not taken into account.

Cytotoxic mechanisms of doxorubicin at clinically relevant concentrations in breast cancer cells.

Doxorubicin (DOX) is a chemotherapeutic agent frequently used for the treatment of a variety of tumor types, such as breast cancer. Despite the long history of DOX, the mechanistic details of its cytotoxic action remain controversial. Rather than one key mechanism of cytotoxic action, DOX is characterized by multiple mechanisms, such as (1) DNA intercalation and adduct formation, (2) topoisomerase II (TopII) poisoning, (3) the generation of free radicals and oxidative stress, and (4) membrane damage through altered sphingolipid metabolism. Many past reviews of DOX cytotoxicity are based on supraclinical concentrations, and several have addressed the concentration dependence of these mechanisms. In addition, most reviews lack a focus on the time dependence of these processes. We aim to update the concentration and time-dependent trends of DOX mechanisms at representative clinical concentrations. Furthermore, attention is placed on DOX behavior in breast cancer cells due to the frequent use of DOX to treat this disease. This review provides insight into the mechanistic pathway(s) of DOX at levels found within patients and establishes the magnitude of effect for each mechanism.

Intracellular trafficking and cytotoxicity of a gelatine-doxorubicin conjugate in two breast cancer cell lines.

Details of intracellular pathways of cytotoxicity remain unclear for doxorubicin conjugates being studied to treat breast cancer tumours. A high molecular weight gelatine-doxorubicin conjugate was investigated with an emphasis on lysosome participation. The conjugate was synthesised and characterised. Cell uptake and cellular localisation in MCF-7 and triple negative breast cancer (TNBC) MDA-MB-231 cells were determined with fluorescence microscopy. Nuclear content of released DOX was determined by UHPLC. Cytotoxicity was determined by the MTT assay. Lysosome membrane permeabilization (LMP) was followed by lysosomal release of fluorescently labelled dextran. After incubation at an equivalent 10 µM DOX, conjugate lysosome accumulation was substantial in both cell lines by 24 h, at which time the conjugate cytotoxic effect was first observed. By 48 h, the conjugate was nearly fourfold more toxic in TNBC than in MCF-7 cells. The MCF-7 nucleus drug content from conjugate released DOX was small but confirmed intra-lysosomal drug release. The conjugate induced LMP in 100% of TNBC cells but LMP was virtually absent in MCF-7 cells. These results suggest that the conjugate induces cytotoxicity by a lysosomal pathway in MDA-MB-231 cells and has potential for treatment of TNBC tumours. Support: NIH/NCI R15CA135421, the Agnes Varis Trust for Women's Health.

Gelatin-methotrexate conjugate microspheres as a potential drug delivery system.

Gelatin-methotrexate microspheres for intra-tumor administration have possibilities for minimizing systemic toxicities of methotrexate (MTX) and overcoming its resistance. Gelatin-MTX conjugates prepared by a carbodiimide reaction were crosslinked with glutaraldehyde to form microspheres (MTX:gelatin molar ratios of 2:1, 15:1, and 21:1). Microspheres were evaluated under in vitro tumor conditions at pH 6.5 and 37 degrees C with and without Cathepsin B (Cat B). Some microspheres were capped with an ethanolamine/cyanoborohydride procedure. SEM of broken microspheres revealed a hollow shell structure. Superficial Cat B degradation influenced some free MTX release but produced no conjugate fragment release. HPLC measured release of fragments (<10 kDa) was very little and release of free MTX was small. However, higher drug load microspheres released less free MTX than lower drug load, a substantial lag phase of free MTX release from capped microspheres changed to an initial rapid release in uncapped microspheres, and fragments were only released from uncapped microspheres. Opened unstable Schiff base crosslinks in uncapped microspheres may allow enzyme to produce conjugate fragments not observed in capped microspheres. Free MTX release may occur from dissolved uncrosslinked conjugate within the hollow microspheres. Important relationships and observations are described that will be useful for gelatin and perhaps other proteinaceous microspheres.

Growth inhibition, drug load, and degradation studies of gelatin/methotrexate conjugates.

Macromolecular gelatin-methotrexate conjugates have potential therapeutic advantages over the free drug. Conjugates with MTX:gelatin molar ratios (MR) ranging from 1:1 to 27:1 were examined for cell growth inhibition, stability, degradation, and methotrexate (MTX) release. Conjugate growth inhibition was less than that of free MTX whose IC(50) value of 1.3 x 10(-8) M was about 10-fold less. Cell uptake of fluorescein labeled gelatin (145 kD) was observed by 24-30 h. Higher MR conjugates produced less growth inhibition, measurably greater stability at pH 7.4 based on MTX release, and had less gelatin degradation in the conjugate by the lysosomal enzyme Cathepsin B (Cat B) compared to low MR conjugates. Cat B conjugate degradation was greater at the in vitro lysosomal pH of 4.8 than the intra-tumor pH of 6.5. The presence of Cat B did not meaningfully affect MTX release, but less MTX was released at pH 4.8 than pH 6.5. The maximum MTX release was a relatively low 7% after 72 h at pH 6.5 for the low MR conjugate. Low molecular weight conjugate fragments were also produced and were also influenced by pH and MR. Reduced growth inhibition by high MR conjugates may be due to a hindered enzymatic degradation in the lysosomes. A strong peptide conjugate bond at lysosomal pH and a 24-30 h delayed gelatin uptake may contribute to reduced growth inhibition of the conjugate compared to free MTX. MTX release under these in vitro conditions occurs by aqueous hydrolysis, not by Cat B cleavage of the conjugate bond.

Crosslinked gelatin matrices: release of a random coil macromolecular solute.

The purpose of this investigation was to evaluate the effect of matrix crosslinking and solute size on release of a random coil macromolecular solute from crosslinked gelatin matrices. Gelatin hydrogel matrices crosslinked with different molar ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC):epsilon-amino groups on gelatin (1:1, 4:1, and 10:1) were prepared containing dextran of molecular weights 12, 20, and 77 kDa, and hydrodynamic diameters 54, 74, and 133 A, respectively. The extent of matrix crosslinking was determined quantitatively and used to calculate the molecular weight between crosslinks (Mc). The Mc parameter and equilibrium swelling ratio (Qm) were used to calculate an estimated matrix mesh size (xi). The in vitro release of incorporated dextran was evaluated at 37 degrees C in PBS at pH 7.4 for approximately 80 h. The one-, four- and 10-fold molar ratios of crosslinking agent EDC yielded 24, 41, and 78% of gelatin matrix crosslinking, respectively. The calculated average matrix mesh size ranged from 338 to 90 A. The effect of matrix crosslinking varied with solute size, from retarding diffusional release of the dextran to completely entrapping it inside the crosslinked matrices. These results support the threshold concept of solute size relative to matrix mesh size for release of a flexible, random coil macromolecular solute from a hydrogel.

Multitemperature stability and degradation characteristics of pergolide mesylate oral liquid.

The stability of pergolide mesylate in an oral aqueous liquid was studied. Stability and solubility data were used to determine the degradation characteristics of the drug in this formulation. Samples were stored in the dark at 35°C, 45°C, and 60°C. At 1, 2, 4, 8, 12, and 16 weeks, samples were removed and stored in a -80°C freezer for high performance liquid chromatography (HPLC) assay at a later date. The initial drug concentration of 0.30 mg/mL was determined by assay after storage at -80°C. A solubility of 6.9 mg/mL was found for pergolide mesylate in the oral liquid at room temperature with a relative standard deviation (RSD) of 4.0%. The degradation process is considered first-order at 25°C and 35°C. At higher temperatures (45°C and 60°C), a color change and curvature at the latter time points in degradation profiles are ascribed to the presence of methylcellulose. The activation energy calculated for degradation of pergolide mesylate in the oral liquid was 21.3 kcal/mol. The time to reach 90% potency (t90) values were calculated to be 43 days and 3 days, respectively, for storage at 25°C and 35°C. Drug concentrations up to ~6 mg/mL can be maintained as a solution at room temperature with this formulation.

The effect of molecular weight, drug load, and charge of gelatin-MTX conjugates on growth inhibition of HL-60 leukemia cells.

PURPOSE: Gelatin-methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells. METHODS: Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometrically. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC50 values were determined on HL-60 cells using a modified MTT assay. RESULTS: IC50 values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC50 values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC50 values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear. CONCLUSIONS: The growth inhibition ranking was MTX>anionic G-MTX-L>anionic G-MTX-H>cationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.