Abstract
Mesoporous vaterite CaCO3 crystals are nowadays one of the most popular vectors for loading of fragile biomolecules like proteins due to biocompatibility, high loading capacity, cost effective and simple loading procedures. However, recent studies reported the reduction of bioactivity for protein encapsulation into the crystals in water due to rather high alkaline pH of about 10.3 caused by the crystal hydrolysis. In this study we have investigated how to retain the bioactivity and control the release rate of the enzyme superoxide dismutase (SOD) loaded into the crystals via co-synthesis. SOD is widely used as an antioxidant in ophthalmology and its formulations with high protein content and activity as well as opportunities for a sustained release are highly desirable. Here we demonstrate that SOD co-synthesis can be done at pH 8.5 in a buffer without affecting crystal morphology. The synthesis in the buffer allows reaching the high loading efficiency of 93%, high SOD content (24 versus 15 w/w % for the synthesis in water), and order of magnitude higher activity compared to the synthesis in water. The enormous SOD concentration into crystals of 10−2 M is caused by the entrapment of SOD aggregates into the crystal pores. The SOD released from crystals at physiologically relevant ionic strength fully retains its bioactivity. As found by fitting the release profiles using zero-order and Baker-Lonsdale models, the SOD release mechanism is governed by both the SOD aggregate dissolution and by the diffusion of SOD molecules thorough the crystal pores. The latest process contributes more in case of the co-synthesis in the buffer because at higher pH (co-synthesis in water) the unfolded SOD molecules aggregate stronger. The release is bi-modal with a burst (ca 30%) followed by a sustained release and a complete release due to the recrystallization of vaterite crystals to non-porous calcite crystals. The mechanism of SOD loading into and release from the crystals as well as perspectives for the use of the crystals for SOD delivery in ophthalmology are discussed. We believe that together with a fundamental understanding of the vaterite-based protein encapsulation and protein release, this study will help to establish a power platform for a mild and effective encapsulation of fragile biomolecules like proteins at bio-friendly conditions.
Highlights
The eye is rather an isolated organ, and the pathological processes within it are preferably treated not via systemic but by local drug intake
Our preliminary studies demonstrated that the activity of superoxide dismutase (SOD) (32 kDa, pI 4.5), significantly decreases when the enzyme is incubated in TRIS buffer solution with the pH above 8.5 (Fig. 1a)
This study shows that SOD can effectively be loaded into CaCO3 vaterite crystals giving extremely high content in the crystals reaching a concentration of up to 380 mg/ml (10−2 M)
Summary
The eye is rather an isolated organ, and the pathological processes within it are preferably treated not via systemic but by local drug intake. Colloids and Surfaces B: Biointerfaces 181 (2019) 437–449 ophthalmic drugs to the eye These include, first and foremost, the use of drug complexes with cyclodextrins [4], the combined use of ophthalmic drugs with agents that enhance the penetration of drugs through biological membranes (EDTA, taurocholic and capric acids) [5], the use of polymer gels [6], colloidal systems containing a drug immobilized on a carrier. Various methods of SOD modification are used to protect the enzyme against adverse environmental effects, to prolong the release and achieve the targeted delivery These methods include: chemical modification (PEGylation [31], binding to lecithin [32]), inclusion in liposomes [33], as well as micro- and nano-encapsulation of the enzyme [18,34,35,36], enzyme immobilization using the layer-by-layer assembly [37]. This study is indispensable for getting fundamental knowledge on encapsulation and controlled release of proteins and other bioactive fragile biomolecules employing the vaterite CaCO3 crystals
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