Table 3:

Biorelevant drug release assays for NDDS

Sr.	Novel biorelevant method	Relevance/Purpose	Reference
1	‘Two-stage reverse dialysis’ method was used to test drug release from Tenofovir liposomes. (stage 1: dialysis in pH 7.4 HEPES buffer solution and, stage 2: dialysis in 1% TX100 solution in HEPES buffer solution)	Stage 1: represented the liposomal drug release into the blood. Stage 2: represented drug release inside targeted tissue.	(Xu et al., 2012)
2	Drug-loaded ‘donor’ large unilamellar vesicles were incubated with ‘acceptors’ multilamellar vesicles. Drug released to the acceptor vesicles was measured (instead of drug release into the release medium).	Multilamellar vesicles mimicked the physiological lipid membrane. Drug loaded donor vesicles showed rapid drug leakage to reflect true in vivo drug retention of liposomes.	(Shabbits et al., 2002)
3	a) ‘Target-sensitive liposomes’ * were developed to test Streptokinase (a thrombolytic drug) drug release. More efficient drug release was found when liposomes were incubated with activated platelets (compared with resting platelets). b) Streptokinase release was also studied in PBS (pH 7.4), using sample and separate method. *having high affinity towards activated platelets- present at the site of clot formation.	a) When target sensitive liposomes were bound to integrin receptors present on activated platelets (target) they got destabilized to release the drug more efficiently. b) Only 40% drug release in 12 h depicted that liposomes could trap the drug effectively with minimum release in systemic circulation on their way to target cells (activated platelets).	(Vaidya et al., 2016)
4	Dissolution/solubility of API was tested in the target media (under relevant hydrodynamic conditions) before dissolution testing from dosage form. It was suggested that stability/ solubility problems can be easily identified using the API alone. Three categories of biorelevant and clinically relevant media were used to support future development of predictive and meaningful in vivo release tests for parenteral liposomal formulations. Amphotericin B (poorly water soluble and highly protein-bound drug) was model drug.	This study emphasized the need to identify suitably discriminating dissolution test conditions (apparatus and hydrodynamic environment) using flow-through cell dissolution test apparatus. Category 1: to test effect of albumin concentration. Category 2: to test effect of biorelevant concentrations of plasma components (bile salts, phospholipids, cholesterol, albumin). Category 3: to test effect of biorelevant or synthetic surfactants with/without albumin (simulated hypoalbuminaemic plasma medium).	(Díaz de León-Ortega et al., 2020).
5	This study postulated that protein corona can affect biodistribution and release behavior of NDDS. Hence, it is important to evaluate the protein-drug interactions. Two different dialysis-based methods (Dispersion releaser technology in conjunction with USP 2 and A4D dialysis adapter with USP 4) were used in combination with a four-step mathematical model. Release medium comprised of PBS at a pH of 7.4 supplemented with 0.1% of ß-CD (solubilizer) and different concentrations (or absence) of fetal calf serum. Dispersion releaser technology was capable to reflect the drug transfer from liposomes to proteins. However, rapid agglomeration of proteins in the fine capillaries of USP 4 altered the hydrodynamics inside the sample cell and could not reflect the conditions defined by media composition.	Four-step mathematical model was used to exclude the impact of membrane pore size** on drug release. Step1: Determination of concentration in donor compartment. Step2: Determination of concentration profile in acceptor compartment (assuming Fick’s law of diffusion). Step3: Determination of drug permeation coefficient (km) using non-linear regression fitting. Step4: Determination of normalized permeation of drug in donor compartment. ** In conventional diffusion methods the release kinetics is affected by both, the membrane pore size and the serum protein binding.	(Wallenwein et al., 2019)
6	Human pharmacokinetics of liposomal temoporfin was predicted using a hybrid in silico model. • Biorelevant release media, comprising PBS (pH 7.4) (0.01% Me-β-CD, 1% Pen-Strep, 10% FBS) was used and liposome stability was investigated using nanoparticle tracking analysis (NTA). • Intracellular uptake was determined into mononuclear phagocytes.• In vitro drug release was investigated with Dispersion releaser (DR) technology using two membrane sizes (MWCO of 50 kDa and 300 kDa). • Four-step model was used to normalize drug release profiles. • Three-parametric reciprocal powered time model (3RPT) was used to describe normalized drug release profiles. • Multicompartment in silico model was developed for rats and humans using Stella® Architect modeling software. Initially, in silico model was based on rat data and later adjusted to human pharmacokinetic parameters by applying allometric scaling. The simulations were verified using plasma concentration–time profiles obtained from phase I clinical trial	• The biorelevant release medium reflected the in vivo dissolution pressure of drug (temoporfin) in blood circulation. • Uptake rate was in line with release behavior for liposomes. MWCO of 50 kDa showed effective retention of serum albumin (molecular weight 67 kDa). • Four-step model excluded the effect of membrane permeation and drug-protein transfer on the release profiles. • 3RPT model (a modified reciprocal powered time model) is based on a combination of diffusion and dissolution processes and can suitably describe the in vitro release profiles of NPs. • Deconvolution of plasma concentration-time profile into different fractions relevant for the in vivo efficacy and safety was achieved. Hybrid in silico model was able to predict liposome in vivo performance in humans.	(Jablonka et al., 2020)
7	Ten conventional release models (zero order, first order, Hixson-Crowell, Higuchi, Power Law etc.) and three novel models, including reciprocal powered time (RPT) model were used to evaluate the release data of 32 drugs from 106 NDDS.	Novel RPT model (along with Weibull and Wagner’s log-probability models) was able to fit the drug release data most accurately from NDDS (nanosphere, nanocapsules, nanocrystal and nanoemulsion).	(Barzegar-Jalali et al., 2008)
8	RPT model was used to describe Chlorpropamide solid dispersions. Drug release was compared with other conventional kinetic models (Power Law, First order, Weibull, Cube-root Law etc.)	Smaller values of mean absolute percent deviation and overall mean percent deviation for RPT model reflected more accurate prediction of fraction of drug dissolved.	(Barzegar-Jalali & Dastmalchi, 2007)
9	The RPT model was successfully applied for kinetic analysis of drug release from Ibuprofen solid dispersions.	RPT model can be applied for diffusion rate limited, dissolution rate limited, and dissolution-diffusion rate limited drug release processes. (justified via unification of the Fick’s first law of diffusion and the Noyes- Whitney law of dissolution).	(Mohammadi et al., 2010)
10	Dispersion releaser technology effectively discriminated among different polymeric (PLGA, PLA, and Eudragit® RS PO) NDDS of flurbiprofen in the presence of biorelevant media. The effect of bovine serum albumin was studied on membrane permeability of flurbiprofen using four-step mathematical model. Two different mathematical models: reciprocal of time (RPT) and the three-parameter model.	Acceptable curve fitting was achieved with both models. RPT (reciprocal powered time) is an easy to apply model. It explains drug release by combination of diffusion and dissolution mechanisms. Three parameter model describes the different mechanisms involved in the release behavior more appropriately. It analyzes the influence of molecular weight of polymeric drug carriers, nanoparticle diameter, and composition of release medium on drug release.	(Janas et al., 2017)