Protein Immobilization on Heterogeneous (SiO2/ZnO) Hollow-Mesoporous Nanocapsules Prepared by Imprinting CPMV: Drug Delivery and Possibility of Immunological Applications.

Herein, we report the protein immobilization and stability studies of heterogeneous hollow mesoporous nanocapsules (Hhmn) for drug and protein delivery. The final results of the diverse precursors such as TEOS, TMOS, APTES, and zinc acetate on the formation of the hollow-mesoporous architecture of nanocapsules have been assimilated in this work. Three types of Hhmn of various sizes were synthesized. Among the three Hhmn, sample I and II nanocapsules were synthesized in the presence of zinc acetate and were identified to be amorphous in nature. Sample III nanocapsules synthesized in the absence of zinc acetate were analogous to the other two synthesized samples. Physiochemical analysis showed the formation of zinc phosphate in the silica matrix for the samples when synthesized with zinc acetate. Specific surface area analysis revealed that sample III has a relatively higher specific surface area. Further, the drug/dye loading and release capacity for the nanocapsules were studied using doxorubicin (DOX) and imatinib mesylate (IM) as model anticancerous drugs and rhodamine 6G as a model dye. Among the synthesized nanocapsules, sample III was shown to have a higher loading capacity for DOX (∼128 µg). From the release kinetic studies of drug/dye, sample III nanocapsules demonstrate a controlled release pattern of DOX and IM. Additionally, protein adsorption and stability studies of samples I and II revealed that the BSA adsorption capacity increases with the increase in the initial concentration of BSA. Furthermore, analysis of the release profiles of BSA and OVA leads to the conclusion that the heterogeneous nanocapsules show a higher loading capacity and sustained release pattern toward OVA. These properties of the nanocapsules highlight their path to immunological applications.

Biomimicked and CPMV-Imprinted Hollow Porous Zinc Phosphate Nanocapsules and Their Therapeutic Efficiency.

Hollow zinc phosphate nanocapsules (hZPNCs) are an alloplastic biomaterial that has been synthesized to deliver chemotherapeutic drugs in a sustained manner. A very simple one-pot synthesis approach has been employed to synthesize hZPNCs by using cowpea mosaic virus (CPMV) in the presence of phosphate buffer (PBS) (0.01 M PBS, pH ∼7.2) with zinc acetate precursor. The synthesis mechanism of hZPNCs relies on the basis of biomineralization, where the precursor molecules initiate mineralization with the help of amino acid residues present on the CPMV capsid. The synthesized hollow nanocapsules were of diameter ∼50-60 nm and porous shell with thickness of ∼4 nm. The cavity performed as a reservoir for the anticancer drugs (DOX and IM). The release kinetic studies show the positive aspect of hZPNCs to be labeled as drug delivery cargo for sustained delivery. In vitro cytotoxic studies of hZPNCs and hZPNCs-chemo drugs on HEK293, HEPG2, and K562 cells were performed. The cytotoxic studies show that hZPNCs-DOX and hZPNCs-IM arrest the cell cycle of carcinoma cells (HEPG2 and K562 cells) at relatively low IC50 and that the inhibition efficiency is dosage dependent. Furthermore, through HRTEM, in vitro cellular interactions of carcinoma cells with hZPNCs and chemo drug-loaded hZPNCs were confirmed by the cryo-sectioning of cells before and after the incubation. These studies revealed the likely endocytic pathway for the nanocapsules entering the cell and executing the specific action of delivering the anticancer drugs. Together, these results reveal the hZPNCs as potential sustained drug delivery agents.

Bio-inspired synthesis of a hierarchical self-assembled zinc phosphate nanostructure in the presence of cowpea mosaic virus: in vitro cell cycle, proliferation and prospects for tissue regeneration.

Self-assembly is an important auto-organization process used in designing structural biomaterials which have the potential capability to heal tissues after traumatic injury. Although various materials having the ability to heal after injury are available, there is still a substantial need to develop new improved materials. To address this issue, we have developed hierarchical three-dimensional (3D) self-assembled zinc phosphate (Zn3(PO4)2) in the presence of cowpea mosaic virus (CPMV). Zn3(PO4)2 nanoparticles are self-assembled into nanosheets with a high degree of isotropy and then self-organized into a 3D structure that can enhance surface interactions with biological entities. The self-assembled structure is formed through the auto-organization of nanoparticles of size ∼50 nm under the influence of CPMV. The cellular response of self-assembled Zn3(PO4)2 and cell-particle adhesion behavior have been investigated through in vitro studies using modeled osteoblast-like MG63 cells. Self-assembled Zn3(PO4)2 resulted in proliferation of MG63 cells of up to 310% within 7 days of incubation. A 15% higher proliferation was obtained than with commercially available hydroxyapatite (HAp). Immunofluorescent analysis of MG63 cells after co-culturing with self-assembled Zn3(PO4)2 confirmed the healthy cytoskeletal organization and dense proliferation of MG63 cells. Further, Zn3(PO4)2 exhibited ∼28% cell-cycle progression in S phase, which is higher than obtained with commercially available HAp. Overall, these results demonstrate the multiple functions of hierarchical self-assembled Zn3(PO4)2 in the regeneration of bone tissue without defects and increasing the formation of cellular networks, and suggest its use in bone tissue engineering.

Mesoporous ZnO nanocapsules for the induction of enhanced antigen-specific immunological responses.

The application of nanotechnology in vaccinology has fuelled rapid advancement towards the design and development of nanovaccines. Nanoparticles have been found to enhance vaccine efficacy through the spatiotemporal orchestration of antigen delivery to secondary lymphoid organs and antigen-presentation by Antigen Presenting Cells (APCs) synchronized with stimulation of innate and adaptive immune responses. Metal based nanoparticles (MNPs) have been extensively engineered for the generation of nanovaccines owing to their intrinsic adjuvant-like properties and immunomodulatory functions. Furthermore, mesoporous nanocapsules of late have attracted researchers due to their precise size and exclusive capacity to encapsulate a wide range of biomolecules and their sustained release at the targeted sites. Herein, we have designed a novel mesoporous ZnO nanocapsule (mZnO) having a size of ∼12 nm with an average pore diameter of 2.5 nm, using a surfactant-free sonochemical method and investigated its immunomodulatory properties by using Ova loaded mZnO nanocapsules [mZnO(Ova)] in a mice model. Our findings show that mZnO(Ova) administration steered the enhanced expansion of antigen-specific T-cells and induction of IFN-γ producing effector CD4+ and CD8+ T-cells. Also, antigen-specific IgG levels were enriched in both the serum and lymph nodes of mZnO(Ova) immunized mice. Further, we noticed a substantial increase in serum IgG2a or IgG2b levels and IFN-γ secretion in Ova restimulated splenocytes from mZnO(Ova) immunized mice, indicating that mZnO(Ova) skew Th1 type immune response. Overall, the uniqueness of mZnO nanocapsules in terms of the defined particle to pore numbers ratio (maximum of three cavities per particle) allows loading antigens efficiently. Given these features in combination with its immunomodulatory characteristics reinforces the idea that mZnO could be used as an effective antigen-adjuvant platform for the development of novel nano-based vaccines against multiple diseases.

CPMV-induced synthesis of hollow mesoporous SiO2 nanocapsules with excellent performance in drug delivery.

Hollow mesoporous-SiO2 nanocapsules have been synthesized at room temperature using unmodified cowpea Mosaic Virus (CPMV) as a template, and without using any catalyst or surfactant during the synthesis. The average size of the capsules synthesized was ∼200-250 nm with a 60-100 nm hollow core. The resulting nanocapsules were characterized using high resolution transmission electron microscopy (HRTEM). The biocompatibility of the hollow mesoporous SiO2 nanocapsules was investigated with an MTT assay using the RAW 264.7 cells, HepG2 cells (human liver carcinoma cells), and Hek293 cells (human embryonic kidney cells). The nanocapsules were loaded with fluorescent molecules (rhodamine 6G), doxorubicin (DOX) ­ an anticancer drug, and chloroquine diphosphate (CQDP) ­ an antimalarial drug, and their release was studied using a UV-Vis spectrometer. The development of surfactant free, bio-safe, hollow and mesoporous SiO2 nanocapsules with CPMV provides a route for the synthesis of porous nanocapsules for drug loading and the sustained delivery of drugs. The synthesis method for hollow mesoporous SiO2 nanocapsules using CPMV is novel, straightforward, and further demonstrates that, in general, nanoformulated capsules can be used for various drug-delivery-based therapeutic applications. To check the in vitro efficacy in medical biotechnology, Hek293 and HepG2 cell lines were used to study the cell viability of DOX-loaded hollow silica nanocapsules. The results show that the bio SiO2 nanocapsules synthesized with CPMV present an effective cargo and are suitable for nanoformulating with DOX, with the resultant nanoformulation showing good promise for killing cancer specific cells.

Facile synthesis of self-assembled spherical and mesoporous dandelion capsules of ZnO: efficient carrier for DNA and anti-cancer drugs.

This work presents a new facile strategy to fabricate self-assembled spherical and mesoporous submicron-sized capsules, 'dandelions', of ZnO nanorods and nanoparticles. Self-assembled 'dandelion' capsules have been synthesized from Zn(Ac)2 and Igepal CO-520, and the mechanistic approach for the growth of self-assembled ZnO capsules has been elucidated. Physical characteristics of the novel capsules responsible for biomedical applications have been studied through XRD, Raman spectroscopy, UV-Vis-NIR spectroscopy, XPS and EPR. The mechanical stability of the capsules has been characterized using high energy ultrasound with time in 10% PBS buffer. The biocompatibility of the capsules has been investigated with a cell-based study using normal lymphocyte and K562 cancer cells through MTT assay. The loading and release efficiency of the fluorescent molecules (Rhodamine 6G), anti-cancer drugs (doxorubicin hydrochloride, DOX), and deoxyribonucleic acid (DNA) have been investigated. All the results indicate the high potential of self-assembled ZnO 'dandelion' capsules in relevant applications, such as for sustained drug delivery with the formation of a {(ZnO)n δ+-(DOX)m} complex and gene delivery with the formation of a {(ZnO)n δ+-(DNA)m complex, in medical biotechnology. The fabrication of such self-assembled idiosyncratic capsules is very simple, feasible, and cost effective; moreover, it demonstrates improved performance in drug and gene delivery applications.