Nanodevices for the immobilization of therapeutic enzymes.

Therapeutic enzymes are one of the most promising applications of this century in the field of pharmaceutics. Biocatalyst properties can be improved by enzyme immobilization on nano-objects, thereby increasing stability and reusability and also enhancing the targeting to specific tissues and cells. Therapeutic biocatalyst-nanodevice complexes will provide new tools for the diagnosis and treatment of old and newly emerging pathologies. Among the advantages of this approach are the wide span and diverse range of possible materials and biocatalysts that promise to make the matrix-enzyme combination a unique modality for therapeutic delivery. This review focuses on the most significant techniques and nanomaterials used for enzyme immobilization such as metallic superparamagnetic, silica, and polymeric and single-enzyme nanoparticles. Finally, a review of the application of these nanodevices to different pathologies and modes of administration is presented. In short, since therapeutic enzymes constitute a highly promising alternative for treating a variety of pathologies more effectively, this review is aimed at providing the comprehensive summary needed to understand and improve this burgeoning area.

Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase.

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. However, in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA) activities were tested in 59% (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 months, meanwhile K-CLEAs kept 51% of their activity. K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment.

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.

Polyvinyl alcohol-pectin cryogel films for controlled release of enrofloxacin.

The release of enrofloxacin entrapped in polyvinyl alcohol (PVA) cryogel at pH 5.5 showed a first-order kinetic, releasing 69.7% of the antibiotic after 4.5 h at 37 °C. In order to slow down the fluoroquinolone release rate, high-methoxylated pectin was added into the cryogel (PVA-P). A film containing 1.0% (w/v) HM pectin and 5.0 µg/ml enrofloxacin released only 3.7% of the antibiotic after 4.5 h. Since the FTIR spectrum showed that most of the interactions between PVA-P matrix and enrofloxacin were due to polar groups (carboxylate and amine), a two-layer film system was designed to modulate the releasing rate of the drug. The top film equilibrated with 0.75 or 1.5 M NaCl release up to 41.9% and 89.0% of the enrofloxacin in 4 h, respectively. The release rate of enrofloxacin was found dependent on NaCl concentration in the upper gel layer. The two-layer cryogel system showed attractive features for transcutaneous antibiotic delivery.