Phospholipid Vesicle-based Permeation Assay Research Articles

Development of a novel human stratum corneum mimetic phospholipid ­vesicle-based permeation assay models for in vitro permeation studies

Objectives To develop and evaluate a novel human stratum corneum (SC) mimetic phospholipid vesicle-based permeation assay (PVPASC) model for in vitro permeation studies. Significance Due to the increasing restrictions on the use of human and animal skins, artificial skin models have attracted substantial interest in pharmaceuticals and cosmetic industries. In this study, a modified PVPASC model containing both SC lipids and proteins was developed. Methods The PVPASC model was optimized by altering the lipid composition and adding keratin in the formulation of large liposomes. The barrier properties were monitored by measuring the electrical resistance (ER) and permeability of Rhodamine B (RB). The modified PVPASC model was characterized in terms of the surface topography, solvent influence and storage stability. The permeation studies of the active components in Compound Nanxing Zhitong Plaster (CNZP) were performed to examine the capability of PVPASC in the application of skin penetration. Results The ER and P app values of RB obtained from the optimized PVPASC model indicated a similar barrier property to porcine ear skin. Scanning electron microscope analysis demonstrated a mimic ‘brick-and-mortar’ structure. The PVPASC model can be stored for three weeks at −20 °C, and withstand the presence of different receptor medium for 24 h. The permeation studies of the active components demonstrated a good correlation (r 2 = 0.9136) of P app values between the drugs’ permeation through the PVPASC model and porcine ear skin. Conclusion Keratin contained composite phospholipid vesicle-based permeation assay models have been proven to be potential skin tools in topical/transdermal permeation studies.

An Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability.

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways.

Predicting Oral Absorption of fenofibrate in Lipid-Based Drug Delivery Systems by Combining In Vitro Lipolysis with the Mucus-PVPA Permeability Model

The aim of this work was to develop a new in vitro lipolysis-permeation model to predict the in vivo absorption of fenofibrate in self-nanoemulsifying drug delivery systems (SNEDDSs). More specifically, the in vitro intestinal lipolysis model was combined with the mucus-PVPA (Phospholipid Vesicle-based Permeation Assay) in vitro permeability model. Biosimilar mucus (BM) was added to the surface of the PVPA barriers to closer simulate the intestinal mucosa. SNEDDSs for which pharmacokinetic data after oral dosing to rats was available in the literature were prepared, and the ability of the SNEDDSs to maintain fenofibrate solubilized during in vitro lipolysis was determined, followed by the assessment of drug permeation across the mucus-PVPA barriers. The amount of drug solubilized over time during in vitro lipolysis did not correlate with the AUC (area under the curve) of the plasma drug concentration curve. However, the AUC of the drug permeated after in vitro lipolysis displayed a good correlation with the in vivo AUC (R2 > 0.9). Thus, it was concluded that the in vitro lipolysis–mucus-PVPA permeation model, simulating the physiological digestion and absorption processes, was able to predict in vivo absorption data, exhibiting great potential for further prediction of in vivo performance of SNEDDSs.

The Vaginal-PVPA: A Vaginal Mucosa-Mimicking In Vitro Permeation Tool for Evaluation of Mucoadhesive Formulations.

Drug administration to the vaginal site has gained increasing attention in past decades, highlighting the need for reliable in vitro methods to assess the performance of novel formulations. To optimize formulations destined for the vaginal site, it is important to evaluate the drug retention within the vagina as well as its permeation across the mucosa, particularly in the presence of vaginal fluids. Herewith, the vaginal-PVPA (Phospholipid Vesicle-based Permeation Assay) in vitro permeability model was validated as a tool to evaluate the permeation of the anti-inflammatory drug ibuprofen from liposomal formulations (i.e., plain and chitosan-coated liposomes). Drug permeation was assessed in the presence and absence of mucus and simulated vaginal fluid (SVF) at pH conditions mimicking both the healthy vaginal premenopausal conditions and vaginal infection/pre-puberty/post-menopause state. The permeation of ibuprofen proved to depend on the type of formulation (i.e., chitosan-coated liposomes exhibited lower drug permeation), the mucoadhesive formulation properties and pH condition. This study highlights both the importance of mucus and SVF in the vaginal model to better understand and predict the in vivo performance of formulations destined for vaginal administration, and the suitability of the vaginal-PVPA model for such investigations.

Impact of Mucin on Drug Diffusion: Development of a Straightforward in Vitro Method for the Determination of Drug Diffusivity in the Presence of Mucin.

Mucosal drug delivery accounts for various administration routes (i.e., oral, vaginal, ocular, pulmonary, etc.) and offers a vast surface for the permeation of drugs. However, the mucus layer which shields and lubricates all mucosal tissues can compromise drugs from reaching the epithelial site, thus affecting their absorption and therapeutic effect. Therefore, the effect of the mucus layer on drug absorption has to be evaluated early in the drug-development phase, prior to in vivo studies. For this reason, we developed a simple, cost-effective and reproducible method employing UV-visible localized spectroscopy for the assessment of the interaction between mucin and drugs with different physicochemical characteristics. The mucin–drug interaction was investigated by measuring the drug relative diffusivity (Drel) in the presence of mucin, and the method was validated by fitting experimental and mathematical data. In vitro permeability studies were also performed using the mucus-covered artificial permeation barrier (mucus–PVPA, Phospholipid Vesicle-based Permeation Assay) for comparison. The obtained results showed that the diffusion of drugs was hampered by the presence of mucin, especially at higher concentrations. This novel method proved to be suitable for the investigation on the extent of mucin–drug interaction and can be successfully used to assess the impact that the mucus layer has on drug absorption.

Development of a novel human stratum corneum model, as a tool in the optimization of drug formulations.

Transdermal delivery represents a very attractive administration route that provides various advantages over other methods of administration, including enhanced patient compliance via non-invasive, painless and simple application and reduced side effects. Thereby, the research on suitable drugs for this route continues to increase. However, most of drug candidates face the challenges of low drug permeability across the skin's biologically active barrier - the stratum corneum (SC). In this context, a low cost, simple screening tool to evaluate penetration of drug candidates in a human SC barrier model was developed. The in vitro model is based on a modified phospholipid vesicle-based permeation assay (PVPA) with a lipid composition close to human SC layer. The new SC PVPA model can be stored up to 2 weeks at -20 °C, withstand a pH range from 2.0 to 8.0 and the presence of co-solvents (DMSO, oleic acid and cremophor®) without losing their integrity. The human mimicking SC PVPA model was able to detect calcein permeability differences when different drugs, applied in the therapy of skin-related diseases, were present. The obtained data correlated well with the well accepted pig ear model, which highlights the potential of this new human SC model.

Mucus-PVPA (mucus Phospholipid Vesicle-based Permeation Assay): An artificial permeability tool for drug screening and formulation development

The mucus layer covering all mucosal surfaces in our body is the first barrier encountered by drugs before their potential absorption through epithelial tissues, and could thus affect the drugs’ permeability and their effectiveness. Therefore, it is of key importance to have in vitro permeability models that can mimic this specific environment. For this purpose, the novel mucus phospholipid vesicle-based permeation assay (mucus-PVPA) has been developed and used for permeability screening of drugs and formulations. The model proved to be stable under the chosen conditions and demonstrated the ability to discriminate between compounds with different chemical structures and properties. Overall, a decrease in drug permeability was found in the presence of mucus on top of the PVPA barriers, as expected. Moreover, mucoadhesive (chitosan-coated) and mucopenetrating (PEGylated) liposomes were investigated in the newly developed model. The mucus-PVPA was able to distinguish between the different liposomal formulations, confirming the penetration potential of the tested formulations and the related drug permeability. The mucus-PVPA model appears to be a promising in vitro tool able to mimic the environment of mucosal tissues, and could therefore be used for further drug permeability screening and formulation development.

Development of phospholipid vesicle-based permeation assay models capable of evaluating percutaneous penetration enhancing effect

Objectives: The phospholipid vesicle-based permeation assay (PVPA) model has recently been introduced as an in vitro model which can mimic stratum corneum (SC) barriers to estimate the skin permeability of drugs. The aim of this study was to evaluate whether the PVPA model was suitable for the evaluation of penetration enhancing effect of skin penetration enhancers (PE).Methods: The PVPA model was optimized by changing the lipid composition of both small liposomes (SL), and large liposomes (LL). The barrier properties of the PVPA model were monitored by electrical resistance and permeability measurement of the fluorescent marker Rhodamine B (RB). Then the permeation studies of the five active compounds with different physicochemical properties, namely, ferulic acid, paeoniflorin, albiflorin, tetrahydrocolumbamine, and tetrahydropalmatine, were performed directly on PVPA model to evaluate the penetration enhancing effect of menthol.Results and discussions: The enhancement ratio (ER) ranking of the five active compounds observed using the optimized PVPA model was in accordance with what observed with Franz diffusion cell device using porcine ear skin. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis of PVPA model and porcine ear skin after treatment with menthol has shown similar mechanism of menthol which perturbs the SC lipid arrangement and extracts the SC lipids.Conclusions: In summary, the optimized PVPA model was used for the first time for the evaluation of the permeation enhancing effect. The optimized PVPA model has shown potential to be applied in a more standardized, cheaper, and ethical way for the screening of PE.

In vitro models to estimate drug penetration through the compromised stratum corneum barrier

Objectives: The phospholipid vesicle-based permeation assay (PVPA) is a recently established in vitro stratum corneum model to estimate the permeability of intact and healthy skin. The aim here was to further evolve this model to mimic the stratum corneum in a compromised skin barrier by reducing the barrier functions in a controlled manner. Methods: To mimic compromised skin barriers, PVPA barriers were prepared with explicitly defined reduced barrier function and compared with literature data from both human and animal skin with compromised barrier properties. Caffeine, diclofenac sodium, chloramphenicol and the hydrophilic marker calcein were tested to compare the PVPA models with established models. Results and discussions: The established PVPA models mimicking the stratum corneum in healthy skin showed good correlation with biological barriers by ranking drugs similar to those ranked by the pig ear skin model and were comparable to literature data on permeation through healthy human skin. The PVPA models provided reproducible and consistent results with a distinction between the barriers mimicking compromised and healthy skin. The trends in increasing drug permeation with an increasing degree of compromised barriers for the model drugs were similar to the literature data from other in vivo and in vitro models. Conclusions: The PVPA models have the potential to provide permeation predictions when investigating drugs or cosmeceuticals intended for various compromised skin conditions and can thus possibly reduce the time and cost of testing as well as the use of animal testing in the early development of drug candidates, drugs and cosmeceuticals.

Biomimetic PVPA in vitro model for estimation of the intestinal drug permeability using fasted and fed state simulated intestinal fluids

A prerequisite for successful oral drug therapy is the drug’s ability to cross the gastrointestinal barrier. Considering the increasing number of new chemical entities in modern drug discovery, reliable and fast in vitro models are required for early and efficient prediction of intestinal permeability. To mimic the intestinal environment, use of biorelevant media may provide valuable information on in vivo drug permeation. The present study aims at improving the novel biomimetic phospholipid vesicle-based permeation assay’s (PVPAbiomimetic) biorelevance by investigating the applicability of the biorelevant media; fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). The FaSSIF and FeSSIF’s influence on the permeability of the model drugs acyclovir, indomethacin, griseofulvin and nadolol was then assessed. The barriers’ robustness in terms of storage stability was also evaluated. The barriers were found to maintain their integrity in presence of FaSSIF and FeSSIF. The model drugs showed changes in permeability in presence of the different simulated intestinal fluids that were in agreement with previous reports. Moreover, the barrier showed improved storage stability by maintaining its integrity for 6months. Altogether, this study moves the PVPAbiomimetic an important step towards a better in vitro permeability model for use in drug development.

Phospholipid Vesicle-Based Permeation Assay and EpiSkin® in Assessment of Drug Therapies Destined for Skin Administration

Cost-effective and efficient methods for permeability screening are crucial during early development of drugs, drug formulations, and cosmeceuticals. Alternatives to animal experiments are impelled for both economical and ethical reasons. The aim of this study was to determine the ability of the phospholipid vesicle-based permeation assay (PVPA) to assess the effect of different formulations on drug permeability and thus establish its utility in formulation development. Three model drugs were tested in solutions and as liposomal formulations. The permeability results for the PVPA models were compared with the results for the reconstructed human skin model, EpiSkin(®). The drugs were ranked based on their estimated penetration potentials, and the results were in accordance with what was expected considering the physicochemical properties of the drugs. PVPAs (E-80, ceramide, cholesterol, cholesteryl sulfate, and palmitic acid) was able to distinguish between drug solutions and liposomal formulations; however, EpiSkin(®) detected only small differences between the drugs in solution and formulations. In contrast with EpiSkin(®), which is limited by a 3-day testing window, PVPA barriers can be stored frozen for up to 2 weeks or even up to 16 months, depending on their compositions. The PVPA models are thus more cost effective and efficient than the EpiSkin(®) model for permeability screening during early drug development.

Development of a Biomimetic Phospholipid Vesicle-based Permeation Assay for the Estimation of Intestinal Drug Permeability

Permeability is a crucial property of orally administered drugs. Therefore, in drug discovery, it is important to employ methods suitable for rapidly and reliably screening the permeability of large numbers of new drug candidates. The phospholipid vesicle-based permeation assay (PVPA), a model consisting of a tight layer of liposomes immobilized on a filter, offers potential advantages unmet by other methods and has been successfully used in permeability testing of novel active substances as well as formulations. In this study, the PVPA was developed into a more robust, biomimetic model by employing a lipid composition matching that of the intestinal permeation barrier and performing the experiments at the more biologically relevant pH 6.2. As expected, positively charged basic compounds demonstrated increased permeability through the negatively charged biomimetic barriers, and the degree of correct classification according to in vivo absorption was comparable between the original PVPA and the biomimetic PVPA. The biomimetic PVPA further proved to be tremendously more robust toward the presence of tensides compared with the original PVPA; this is a promising finding that renders the biomimetic PVPA an enhanced ability to estimate the permeability of poorly soluble compounds. Hence, the PVPA model developed in this study has evolved an important step forward.

Liposomes for (trans)dermal drug delivery: the skin-PVPA as a novel in vitro stratum corneum model in formulation development

Penetration potential of vesicles destined for trans(dermal) administration remains to be of great interests both in respect to drug therapy and cosmetic treatment. This study investigated the applicability of the phospholipid vesicle-based permeation assay (PVPA) as a novel in vitro skin barrier model for screening purposes in preformulation studies. Various classes of liposomes containing hydrophilic model drug were examined, including conventional liposomes (CLs), deformable liposomes (DLs) and propylene glycol liposomes (PGLs). The size, surface charge, membrane deformability and entrapment efficiency were found to be affected by the vesicle lipid concentration, the presence of the surfactant and propylene glycol. All liposomes exhibited prolonged drug release profiles with an initial burst effect followed by a slower release phase. The permeation of the drug from all of the tested liposomes, as assessed with the mimicked stratum corneum – PVPA model, was significantly enhanced as compared to the permeability of the drug in solution form. Although the DLs and the PGLs exhibited almost the same membrane elasticity, the permeability of the drug delivered by PGLs was higher (6.2 × 10−6 cm/s) than DLs (5.5 × 10−6 cm/s). Therefore, this study confirmed both the potential of liposomes as vesicles in trans(dermal) delivery and potential of the newly developed skin-PVPA for the screening and optimization of liposomes at the early preformulation stage.

Improved Permeability of Acyclovir: Optimization of Mucoadhesive Liposomes Using the Phospholipid Vesicle-Based Permeation Assay

The antiviral drug acyclovir (ACV) suffers from poor solubility both in lipophilic and hydrophilic environment, leading to low and highly variable bioavailability. To overcome these limitations, this study aimed at designing mucoadhesive ACV-containing liposomes to improve its permeability. Liposomes were prepared from egg phosphatidylcholine (E-PC) and E-PC/egg phosphatidylglycerol (E-PC/E-PG) and their surfaces coated with Carbopol. All liposomal formulations were fully characterized and for the first time the phospholipid vesicle-based permeation assay (PVPA) was used for testing in vitro permeability of drug from mucoadhesive liposome formulations. The negatively charged E-PC/E-PG liposomes could encapsulate more ACV than neutral E-PC liposomes. Coating with Carbopol increased the entrapment in the neutral E-PC liposomes. The incorporation of ACV into liposomes exhibited significant increase in its in vitro permeability, compared with its aqueous solution. The neutral E-PC liposomal formulations exhibited higher ACV permeability values compared with charged E-PC/E-PG formulations. Coating with Carbopol significantly enhanced the permeability from the E-PC/E-PG liposomes, as well as sonicated E-PC liposomes, which showed the highest permeability of all tested formulations. The increased permeability was according to the formulations' mucoadhesive properties. This indicates that the PVPA is suitable to distinguish between permeability of ACV from different mucoadhesive liposome formulations developed for various routes of administration.

Characterization and evaluation of a modified PVPA barrier in comparison to Caco-2 cell monolayers for combined dissolution and permeation testing

Aim of this study was to implement a modified phospholipid vesicle-based permeation assay (PVPA) barrier as alternative to Caco-2 cell monolayers in a combined dissolution and permeation system for testing of solid dosage forms. Commercially available Transwell® inserts were coated with egg phospholipids (Lipoid E 80) and characterized by confocal Raman microscopy. The modified PVPA barrier was then evaluated in permeation studies with solutions of different drugs as well as in combined dissolution and permeation studies utilizing an immediate and an extended release tablet formulation.Raman cross section images demonstrated complete filling of the membrane pores with lipids and the formation of a continuous lipid layer of increasing thickness on top of the membrane during the stepwise coating procedure. Furthermore, it could be shown that this lipid coating remains intact for at least 18h under dynamic flow conditions, significantly exceeding the viability of Caco-2 cell monolayers.Permeability data for both drug solutions as well as for a fast and slow release tablet formulation were in excellent correlation with those data obtained for Caco-2 cell monolayers. Especially under the dynamic flow conditions prevailing in such a setup, the modified PVPA barrier is more robust and easier to handle than epithelial cell monolayers and can be prepared rather easily at a fraction of costs and time. The modified PVPA barrier may therefore represent a valuable alternative to Caco-2 cell monolayers in such context.

New Applications of Phospholipid Vesicle-Based Permeation Assay: Permeation Model Mimicking Skin Barrier

The phospholipid vesicle-based permeation assay (PVPA), based on a tight barrier composed of liposomes mimicking cells, is providing an opportunity to predict passive drug permeability through biological membranes. Although it was originally developed to mimic the intestinal epithelia, this study focuses on its potential as a simple and affordable skin model for transdermal permeation of drug candidates and evaluation of various drugs and formulations at an early development stage. The changes induced in lipid composition of the lipid-based barriers to better mimic the in vivo stratum corneum lipid composition required optimization of liposomal properties and manufacturing conditions applied in barrier formation. The preparation conditions could be modified to prepare lipid-based barriers of different degrees of leakiness, potentially representing different degree of intact and compromised skin. The different PVPA models developed in this study appeared to be able to distinguish between drugs with different degrees of lipophilicity and penetration potential. Moreover, the PVPA can be produced in controlled and reproducible manner with different degree of leakiness. The model could therefore be applied in both pharmaceutical and cosmeceuticals manufacturing and also has the potential to provide deeper insight on safety of nanodelivery systems administered onto the skin.

Application of simulated intestinal fluid on the phospholipid vesicle-based drug permeation assay

The compatibility of fasted state simulated intestinal fluid (FaSSIF) in drug permeation studies employing the phospholipid vesicle-based permeation assay (PVPA) model was confirmed by a set of different integrity indicators. Neither calcein permeability nor electrical resistance were found significantly changed indicating unaffected barrier tightness. Furthermore, the release of phospholipid from the barriers in contact with FaSSIF was negligible, although sodium taurocholate disappeared from the donor – possibly due to transfer into the barrier. Visual examination of the barrier structure by confocal laser scanning microscopy (CLSM) revealed no changes. The model drugs, cimetidine, nadolol, ketoprofen and griseofulvin showed either slightly enhanced or unchanged permeability values in the presence of FaSSIF. This may be attributed to micellar encapsulation and/or slight changes in barrier characteristics. Particularly for poorly soluble drugs, FaSSIF appeared favourable in terms of markedly improved recovery. Moreover, utilisation of BSA in the receiver compartment seems to augment this beneficial effect on recovery rate. It is likely that this experimental set-up affords better sink conditions in the receiver phase, which results in higher fluxes. Overall, a combination of FaSSIF in the donor phase and BSA in the receiver phase facilitates improved experimental output.

In-vitro permeability of poorly water soluble drugs in the phospholipid vesicle-based permeation assay: the influence of nonionic surfactants

The aim of this study was to determine the influence of nonionic surfactants on drug permeability using the phospholipid vesicle-based permeation assay (PVPA), which excludes other than trans-membrane diffusion pathways. Barrier integrity was monitored both by electrical resistance and permeability measurement of the hydrophilic marker calcein. Permeability of the model drugs ketoprofen and nadolol across the PVPA-barrier was measured by HPLC-UV. Micelle association of the model drugs was determined using ultrafiltration, whereby micelle-bound drug and molecular drug were separated. The nonionic surfactant poloxamer 188 was demonstrated not to affect barrier integrity. Drug permeability was found depressed in the presence of poloxamer 188 in a concentration-dependent manner. Both drugs were found to associate with poloxamer 188 micelles. The extent of the decrease in permeability correlated mostly, but not in all cases, with the fraction of micelle-bound drug. Micelle association was one important but not the only factor affecting drug permeability across the PVPA-barrier.

The Phospholipid Vesicle-Based Drug Permeability Assay: 5. Development toward an Automated Procedure for High-Throughput Permeability Screening

In vitro screening for oral absorption has become an essential part of drug discovery and development. Recently, a new phospholipid vesicle-based permeation assay was developed which has shown to satisfyingly predict passive absorption of drugs in humans. The purpose of the current study was to investigate whether the assay may be further developed into a high-throughput tool by automating its most time-consuming steps. The following challenges were addressed: (1) to design, build, and test a heat-sealing machine for mounting of the desired type of filter support onto both single wells and 24-well titer plate inserts and (2) to transfer the permeability assay to a robotic workstation with attached ultraviolet (UV) reader. The workstation is able to pipette and transport both plates and filter inserts and perform on-line photometric quantification of the amount of drug permeated. To enable the robot to move single (Standard Transwell; Corning Inc, Lowell, MA) filter inserts, an extension of the gripping arm was designed, built, and tested. Furthermore, in an alternative approach 24-well filter plates (Millicell; Millipore, Billerica, MA) were used instead of single filter inserts. The latter turned out to be more suitable in terms of error-free high-throughput robotic handling. The permeability values of drugs gained by the two automated procedures were compared with those measured by manual handling of the assay. Only neglectable differences in permeability values were seen. In conclusion, the most time-consuming steps of the assay were shown to be eligible for automation. This represents an interesting addition to the toolbox of in vitro permeability screening assays running in a medium- to high-throughput format due to its easiness, its transferability to other laboratories, and its good correlation with in vivo data on fraction absorbed of drugs in humans.