In vitro and in vivo characterization of Nanoparticles
For in-vitro characterization, NPs may be assayed in biological matrices such as blood, plasma, cells, or primary culture. Some in vitro tests that may be performed include
Drug entrapment efficiency: To find the drug entrapment efficiency solid nanoparticles are solubilized with proper solvent and the drug is separated from the supernatant containing excess by ultra-centrifugation. One ml of the supernatant is withdrawn and suitably diluted with proper solvent. The absorbance of the solution was measured at desired wavelength spectrophotometrically and the drug concentration in the supernatant was obtained by comparison with the analytical curve previously prepared. The amount of drug entrapped in the nanoparticles was obtained by subtracting the quantity of drug in the supernatant liquid from the total amount used for the preparation
In-Vitro drug release studies: The in-vitro drug release study of the prepared drug loaded nanoparticles was performed using the dialysis bag diffusion technique or dissolution technique. In case of diffusion cell analysis the prepared nanosuspension was placed in a dialysis bag, and dropped into beaker containing specific volume diffusion medium maintained at 37C under continuous magnetic stirring. At fixed time intervals the sample of the receptor medium was withdrawn and replaced with fresh solvent media. One ml of sample was withdrawn at periodic time intervals. The samples were analyzed spectrophotometrically at particular wavelength. The percentage of drug released at various time intervals were calculated from the calibration curve constructed previously
Fig 1:Diffusion cell for dissolution studies
For dissolution studies the sample are mainly kept in different dissolution apparatus USP I and USP II. At fixed time intervals the sample is withdrawn for UV and HPLC analysis and the sample volumes to be replaced with fresh solvent media.
Sterility check (e.g., testing for the presence of bacteria, virus or mycoplasma)
Sterility is an important for both in vitro and in vivo application of nanoparticles for various drug and gene delivery. For clinical treatment of the patients parenteral drug have to meet the pharmacopoeia requirement of high degree of purity. The chemical or physical stability of the polymer matrix usually limits most conventional methods for obtaining acceptable sterile products. The drug loaded nanoparticles were evaluated for its sterility by sterility testing as per different Pharmacopeia. The method involves the membrane filtration technology followed by incubation of membrane in culture media containing nutrients broth of Soya bean casein digest medium (SCDM) and Fluid thioglycollate medium (FTM). After filtration of the solution, the membrane was rinsed 3 times with sterile peptone solution. The membrane was then separated cut into two halves. One half of the filter paper was kept into the container with SCDM and the other half was kept into the container with thioglycollate medium. SCDM containers were then incubated at room temperature and FTM containers at 32.5+-2.50C. The containers were monitored for any turbidity or appearance of growth of microorganisms for 14 days. Positive control and negative control tests were also executed to validate the sterility testing procedure.
* Blood contact properties: Blood nanoparticles interaction is an important characteristic to evaluate. The following parameters like plasma protein binding, hemolysis, coagulation, complement activation, cytotoxic activity of NK cells
* Cell uptake and distribution studies include cell binding, NP internalization and receptor targeting.
* Toxicity studies including phase I/II enzyme induction or suppression studies, oxidative stress, apoptosis testing and also necrosis testing. The disadvantages of in vitro testing is that amount of information obtained is limited. In addition, in-vitro testing for biomedical/clinical applications (e.g., therapeutics, in vivo diagnostics) has to be tested in animal models. In vivo assays can supply essential information regarding what may happen when the NPs are inside the body. Some in vivo tests which are important in nanoparticles analysis include dose-response; biodistribution, acute and multi dose safety and efficacy, administration route determination; and absorption, distribution, metabolism, and excretion (ADME). The ultimate goal of in vitro and in vivo testing is to match the physicochemical parameters of the NP to its biological function