2.5. Characterization of polymeric nanoparticles

Chemical integrity between PLGA, drug and other excipient (PVA) of the NPs was determined by identifying specific bond vibration of each ingredient using FTIR spectroscopy (model: Nicolet iS10, Thermo Fisher Scientific, USA). The samples were scanned in the wavenumber range from 400 to 4000 cm⁻¹.

The structure alteration of materials due to heat exchange was measured in order to determine the integrity of the NPs. DSC (Perkin Elmer Pyris Diamond) was used to measure heat exchange capacity in a controlled inert environment at heat rate 10°C min⁻¹ in a temperature range of 5°C to 350°C under constant N₂ flow of 40 ml min⁻¹ [27].

X-ray diffraction of excipients indicates crystallinity which helps to identify the physical state of the compounds. The physical state of pure drug, PLGA and NPs was evaluated using Ultima III (Rigaku) X-ray diffractometer (XRD) with copper target and K-beta filter. Powder sample was mounted on a quartz plate and smoothed to a level surface. The XRD pattern of each sample was measured over an angular range from 5° to 90° (diffraction angle 2θ) in stepwise increments of 0.05° and a scanning speed of 3° min⁻¹ radiation.

Pure TLM was dissolved in methanol (HPLC grade) and was infused in mass spectrometer for Q1 scan. Extracted TLM from NPs was also injected into LC-MS/MS to compare with pure drug. Quantification of TLM in NPs was determined by LC-MS/MS to evaluate encapsulation efficiency of NPs, drug loading and in vitro drug release kinetics.

The physical structure and surface morphology of the NPs were observed with the help of scanning electron microscopy (SEM, EVO 18, Special Edition, Zeiss). The NPs were mounted on an aluminium dais using carbon adhesive. A thin film of gold was spotted over the samples in high vacuum under low pressure using a Quorum Q150T ES.

The physical dimension (particle size), surface potential (zeta potential) and size distribution of NPs were measured by dynamic light scattering technique using a Zetasizer (Nano-ZS90, Malvern Instruments, UK). A small amount (0.1%) of samples (NPs) was dispersed properly in deionized water and poured into a cuvette for analysis [28].

The quantification of drug into NPs was performed using a validated method in LC-MS/MS. The encapsulated drug was separated from the polymer (PLGA) matrix by the following method and introduced in LC-MS/MS to determine drug content of NPs. NPs (1 mg) were dissolved in DCM to extract the encapsulated TLM. Dissolved PLGA was separated by centrifugation at 12 000 r.p.m. after precipitation with 2 ml of methanol. The supernatant solution was collected and dried under N₂ atmosphere at 45°C. The dried sample was reconstituted with a solvent mixture of methanol : water in 1 : 1 ratio. The reconstituted samples were poured into auto-sampler vial for quantification. Entrapment efficiency (%) and drug loading (%) were calculated using equations (2.2) and (2.3) respectively as described earlier [29].

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