Biodegradable Calcium Phosphate Nanoparticles Research Articles

Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and −25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 μg of protein. Group I ducks received 3762 μg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

A Precipitation-Enhanced Emission (PEE) Strategy for Increasing the Brightness and Reducing the Liver Retention of NIR-II Fluorophores.

The lack of organic fluorophores with high quantum yields (QYs) and low liver retention in the second near-infrared (NIR-II) window has become a bottleneck in the bioimaging field. An approach to address these problems is proposed by encapsulating phosphorylated fluorescent dyes into biodegradable calcium phosphate nanoparticles. First, an NIR-II molecule, LJ-2P, is designed with increased water solubility by introducing two phosphate groups. Meanwhile, LJ-2P co-precipitates with calcium ions to form LJ-2P nanoparticles (NPs). The QYs of LJ-2P NPs in aqueous solution is increased by 36.57-fold to 5.12% compared with that of LJ-2P. This unique phenomenon is named as precipitation-enhanced emission (PEE), whose detailed mechanism is explored by femtosecond transient absorption. It is demonstrated that co-precipitation of LJ-2P with calcium ions changes the micro-environment, which restricts the molecular rotation and reduces the interaction of water molecules, especially the excited-state proton transfer. In addition, due to the pH-sensitive nature, more than 80% of the LJ-2P NPs are metabolized in the liver within 24 h. Based on the excellent optical properties and good biocompatibility, high-contrast vascular visualization and breast tumor detecting are achieved. This strategy can apply to other NIR-II fluorophores to achieve high QYs and low liver retention.

Bio-inspired negatively-charged calcium phosphate nanocarriers for cardiac delivery of therapeutic molecules

Event Abstract Back to Event Bio-inspired negatively-charged calcium phosphate nanocarriers for cardiac delivery of therapeutic molecules Michele Iafisco1, Vittoria Di Mauro2, 3, Nicolò Salvarani2, 3, Gloria Belén Ramírez-Rodríguez1, Tatiana Patrício1, Michele Miragoli2, 3, Daniele Catalucci2, 3 and Anna Tampieri1 1 National Research Council (CNR), Institute of Science and Technology for Ceramics (ISTEC), Italy 2 Humanitas Clinical and Research Center, Italy 3 National Research Council (CNR), Institute of Genetics and Biomedical Research (IRGB), Italy Cardiovascular disease (CVD) is a worldwide growing problem that afflicts close to 1% of the population and causes 17.3 million annual premature deaths. Causes include ischemic, toxic, genetic, post-inflammatory, and structural defects, overall resulting in a mortality of ~50% within 5 years from diagnosis[1]. Clinical management of CVDs has improved during the last decades, but despite significant advancements, these pathologies still lead to a poor quality of life and reduced longevity. Therefore, a great challenge is the identification of innovative therapeutic compounds and the development of more efficient, safe, and “patient-friendly” drug-delivery systems. Nanoparticles (NPs) hold great promise for nanomedical systems and provide an alternative strategy for more efficient, controlled, and safe approaches of drug delivery[2]. While largely investigated in the cancer field, the development and use of efficient NPs for the treatment of CVDs is still in its infancy. In fact, to the best of our knowledge, only liposomes or NPs based on synthetic polymers have been investigated so far for the delivery of various therapeutic molecules to myocardial cells[3],[4]. Their use is drastically limited due to the lack of biodegradability and biocompatibility of these kinds of nano-carriers, the uncontrolled drug release in the bloodstream, the poor encapsulation efficacy, and the poor stability. In summary, there is a strong need for the identification of new formulations based on biocompatible and biodegradable NPs to overcome the limitations of NPs currently used for CVD treatment. In this study, novel biocompatible and bioresorbable negatively charged biodegradable calcium phosphate nanoparticles (CaP-NPs) were developed as an innovative therapeutic system for the delivery of bioactive molecules to the heart. Synthesis of CaP-NPs was performed according to a new, straightforward one-pot inspired biomineralization protocol employing citrate as a stabilizing agent and regulator of crystal growth. Via the classical clathrin-mediated path, CaP-NPs were shown to efficiently internalize into HL-1 cardiac cells without promoting toxicity. Additionally, acute and chronic administration of CaP-NPs did not interfere with Ca2+ handling properties and electrophysiological features of HL-1 cells and adult cardiomyocytes. Finally, preliminary experiments validated that CaP-NPs were successfully able to encapsulate bioactive synthetic microRNAs and mimetic peptides, which were efficiently delivered into cardiac cell line in vitro and surprisingly even into cardiac tissue when admistered in vivo. To sum up, our results showed that the CaP-NPs generated in this work are safe and effective drug-delivery systems for a new potential therapeutic treatment of polarized cells such as cardiomyocytes. The Italian Flagship Project NANOMAX – miRnano (Progetto Bandiera MIUR PNR 2011–2013)

Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection

Retroviral infections e.g. HIV still represent a unique burden in the field of vaccine research. A common challenge in vaccine design is to find formulations that create appropriate immune responses to protect against and/or control the given pathogen. Nanoparticles have been considered to be ideal vaccination vehicles that mimic invading pathogens. In this study, we present biodegradable calcium phosphate (CaP) nanoparticles, functionalized with CpG and retroviral T cell epitopes of Friend virus (FV) as excellent vaccine delivery system. CaP nanoparticles strongly increased antigen delivery to antigen-presenting cells to elicit a highly efficient T cell-mediated immune response against retroviral FV infection. Moreover, single-shot immunization of chronically FV-infected mice with functionalized CaP nanoparticles efficiently reactivated effector T cells which led to a significant decrease in viral loads. Thus, our findings clearly indicate that a nanoparticle-based peptide immunization is a promising approach to improve antiretroviral vaccination. From the Clinical EditorIn this study, biodegradable calcium phosphate nanoparticles were used as a vaccine delivery system after functionalization with CpG and Friend virus-derived T-cell epitopes. This vaccination strategy resulted in increased T-cell mediated immune response even in chronically infected mice, providing a promising approach to the development of clinically useful antiretroviral vaccination strategies.

Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity.

Quantitative determination of the composition of multi-shell calcium phosphate–oligonucleotide nanoparticles and their application for the activation of dendritic cells

Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic–polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro.

Biodegradable calcium phosphate nanoparticles as a new vehicle for delivery of a potential ocular hypotensive agent.

The purpose of this study was to determine the efficacy of a newly prepared formulation containing biodegradable calcium phosphate nanoparticles (CAP) and 7-hydroxy-2-dipropyl-aminotetralin (7-OH-DPAT) in pigmented and non-pigmented rabbits using the surrogate end points of intraocular pressure (IOP) and aqueous flow rate. IOP (mmHg) was measured by utilizing a manometrically calibrated Mentor pneumatonometer. Rates of aqueous humor flow were measured with a Fluorotron Master by estimating the dilution rate of fluorescein. In non-pigmented rabbits, the ocular hypotension induced by topical administration of 7-OH-DPAT (75 microg) with CAP (115 microg) was more pronounced and sustained than that of 7-OH-DPAT without CAP. Furthermore, IOP-lowering effects of topically administered 7-OH-DPAT (125 microg) alone were markedly diminished in pigmented rabbits compared to non-pigmented rabbits. However, topical application of 7-OH-DPAT formulated with CAP produced significant dose-related (37.5, 75, 125 microg) reductions of IOP accompanied by suppression of aqueous humor flow rates in pigmented rabbits. It is postulated that 7-OH-DPAT in vehicle without CAP binds to pigments in the anterior segment of the pigmented rabbit's eyes, and this binding limits the 7-OH-DPAT's action. Pretreatment with raclopride, a dopamine D2/D3 receptor antagonist, reduced the ocular hypotensive effect induced by 7-OH-DPAT in vehicle containing CAP thereby supporting the role for dopamine D2/D3 receptors in modulating IOP. It is concluded that CAP, as a delivery system, enhances activity by 7-OH-DPAT in pigmented rabbit eyes suggesting that CAP is potentially useful for achieving controlled and targeted drug delivery for treatment of ocular diseases.