Extracellular vesicles enhance the targeted delivery of immunogenic oncolytic adenovirus and paclitaxel in immunocompetent mice.

Extracellular vesicles (EVs), are naturally occurring cargo delivery tools with the potential to be used as drug vehicles of single agents or combination therapies. We previously demonstrated that human lung cancer cell-derived EVs could be used for the systemic delivery of oncolytic virus (OVs) and chemotherapy drugs such as paclitaxel (PTX), leading to enhanced anti-tumor effects in nude mice. In the current work, we evaluated the biodistribution of EVs by using bioluminescence and fluorescence imaging technologies, thus proving the ability of these EVs-formulations to specifically target the neoplasia, while leaving other body tissues unaffected. Moreover, in vivo imaging of NFκB activation in an immunocompetent reporter mouse model allowed to demonstrate the selective ability of EVs to induce tumor-associated inflammatory reactions, which are characterized by immunogenic cell death and CD3+/CD4+/CD8+ T-cell infiltration. While EVs have the potential to induce a systemic immune reaction by pro-inflammatory cytokines, our study provides compelling evidences of a localized inflammatory effect in the peritumoral area. Collectively, our findings strongly support the systemic administration of EVs formulations with OVs alone or in combination with chemotherapy agents as a novel strategy aimed at treating primary and metastatic cancers.

FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel.

In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti-cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel-containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real-time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next-generation targeted drug delivery systems.

Antitumor effect of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment.

Standard of care for cancer is commonly a combination of surgery with radiotherapy or chemoradiotherapy. However, in some advanced cancer patients this approach might still remaininefficient and may cause many side effects, including severe complications and even death. Oncolytic viruses exhibit different anti-cancer mechanisms compared with conventional therapies, allowing the possibility for improved effect in cancer therapy. Chemotherapeutics combined with oncolytic viruses exhibit stronger cytotoxic responses and oncolysis. Here, we have investigated the systemic delivery of the oncolytic adenovirus and paclitaxel encapsulated in extracellular vesicles (EV) formulation that, in vitro, significantly increased the transduction ratio and the infectious titer when compared with the virus and paclitaxel alone. We demonstrated that the obtained EV formulation reduced the in vivo tumor growth in animal xenograft model of human lung cancer. Indeed, we found that combined treatment of oncolytic adenovirus and paclitaxel encapsulated in EV has enhanced anticancer effects both in vitro and in vivo in lung cancer models. Transcriptomic comparison carried out on the explanted xenografts from the different treatment groups revealed that only 5.3% of the differentially expressed genes were overlapping indicating that a de novo genetic program is triggered by the presence of the encapsulated paclitaxel: this novel genetic program might be responsible of the observed enhanced antitumor effect. Our work provides a promising approach combining anticancer drugs and viral therapies by intravenous EV delivery as a strategy for the lung cancer treatment.

Biological reference materials for extracellular vesicle studies.

Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.

Nanofibrillar cellulose wound dressing in skin graft donor site treatment.

BACKGROUND: Although new therapeutic approaches for burn treatment have made progress, there is still need for better methods to enhance wound healing and recovery especially in severely burned patients. Nanofibrillar cellulose (NFC) has gained attention due to its renewable nature, good biocompatibility and excellent physical properties that are of importance for a range of applications in pharmaceutical and biomedical fields. In the present study, we investigated the potential of a wood based NFC wound dressing in a clinical trial on burn patients. Previously, we have investigated NFC as a topical functionalized wound dressing that contributes to improve wound healing in mice. METHODS: Wood based NFC wound dressing was tested in split-thickness skin graft donor site treatment for nine burn patients in clinical trials at Helsinki Burn Centre. NFC dressing was applied to split thickness skin graft donor sites. The dressing gradually dehydrated and attached to donor site during the first days. During the clinical trials, physical and mechanical properties of NFC wound dressing were optimized by changing its composition. From patient 5 forward, NFC dressing was compared to commercial lactocapromer dressing, Suprathel® (PMI Polymedics, Germany). RESULTS: Epithelialization of the NFC dressing-covered donor site was faster in comparison to Suprathel®. Healthy epithelialized skin was revealed under the detached NFC dressing. NFC dressing self-detached after 11-21days for patients 1-9, while Suprathel® self-detached after 16-28days for patients 5-9. In comparison studies with patients 5-9, NFC dressing self-detached on average 4days earlier compared with Suprathel®. Lower NFC content in the material was evaluated to influence the enhanced pliability of the dressing and attachment to the wound bed. No allergic reaction or inflammatory response to NFC was observed. NFC dressing did not cause more pain for patients than the traditional methods to treat the skin graft donor sites. CONCLUSION: Based on the preliminary clinical data, NFC dressing seems to be promising for skin graft donor site treatment since it is biocompatible, attaches easily to wound bed, and remains in place until donor site has renewed. It also detaches from the epithelialized skin by itself.

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes.

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80 nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80 nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100 nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.

Pharmacokinetic simulations to explore dissolution criteria of BCS I and III biowaivers with and without MDR-1 efflux transporter.

In this study, a pharmacokinetic simulation model was used to explore the dissolution acceptance criteria for BCS I and III biowaivers and to examine the risk of MDR-1 efflux transporter on bioequivalence of substrates. The compartmental absorption and transit (CAT) model with one- or two systemic compartments was used. The parameter values used in the simulations were based on the pharmacokinetics of existing 70 BCS I and III drugs. Based on the simulations BCS I drug products with Tmax of >0.9 h, both dissolution criteria "very rapid" and "rapid and similar" were acceptable. For rapidly absorbed and distributed BCS I drug products with Tmax of 0.6-0.9 h, the dissolution criterion "very rapid" is preferred. If Tmax is less than 0.6 h there is a risk of bioinequivalence for the BCS I drug products regardless of the dissolution criteria. Based on the simulations, all BCS III drug products were good biowaiver candidates with both dissolution criteria. Almost all the BCS III drug products (>89%) and many BCS I products (9-57%) showed risks of bioinequivalence, if an excipient in either product inhibits MDR1-efflux transport of the drug. To eliminate these risks excipients with prior use in bioequivalent products should be used for MDR-1 efflux substrates.

Very rapid dissolution is not needed to guarantee bioequivalence for biopharmaceutics classification system (BCS) I drugs.

Currently, the EMEA, FDA, and WHO as regulatory authorities accept rapidly dissolving (>85% dissolved in 30 min) biopharmaceutics classification system (BCS) I drug products for biowaiver candidates. In the draft EMEA guideline the requirement has been set tighter, that is, the drug product should be very rapidly dissolving (>85% dissolved in 15 min) to be eligible for a biowaiver. Pharmacokinetic modeling of 32 BCS I drugs was performed to demonstrate that very rapid dissolution is not necessary to guarantee bioequivalence for them. Rapid dissolution and similar dissolution profiles are sufficient criteria for all BCS I drugs.

Kinetic simulation model of protein secretion and accumulation in the cell microcapsules.

BACKGROUND: In cell therapy, microencapsulated cells secrete therapeutic protein, which is further released from the microcapsules. In principle, some secreted, but unreleased, protein may accumulate in the microcapsules. The kinetic simulation model was built to simulate the potential accumulation of the protein in the microcapsules. METHODS: The alginate microcapsules were cross-linked with divalent cations to encapsulate either flourescein isothiocyanate (FITC)-dextrans (molecular weights = 4.3, 10.5, 43 kDa) or retinal pigment epithelial cells (ARPE-19). The cells were genetically engineered to produce secreted alkaline phosphatase (SEAP). SEAP production from the cells was quantified with and without microcapsulation and, finally, the cells were killed with toxin to quantify the secreted but yet unreleased SEAP from the microcapsules. The empirical three-compartment kinetic model was constructed based on the release of FITC-dextrans of different molecular weights from the alginate microcapsules with different pore sizes. Protein secretion from the cells into the microcapsules was added to the model. The impact of the microcapsule wall permeability on the steady-state amounts of secreted protein in the microcapsules and in the hypothetical target compartment in the body was simulated. The simulations were compared to the experimental data from the microencapsulated SEAP secreting ARPE-19 cells. RESULTS: The model and the data show that substantial amounts (10-15 daily doses) of protein may accumulate in the microcapsules with poor wall permeability. At high permeability, the accumulation was insignificant. The pharmacokinetic simulations show that even a 1.5-fold increase in the wall permeability may result in a substantial peak in the drug amount in the target compartment, especially if the elimination rate of the protein is high. CONCLUSIONS: The kinetic simulation model for protein secretion from microcapsulated cells is a useful tool for the early kinetic prediction and risk assessment of cell therapy.

Pharmacokinetic simulation of biowaiver criteria: the effects of gastric emptying, dissolution, absorption and elimination rates.

In vitro dissolution tests can be used to waive in vivo bioequivalency studies (biowaiver), if drug has high solubility and high permeability according to biopharmaceutics classification system (BCS I). Then absorption of BCS I drugs is not dependent on drug dissolution or gastrointestinal transit time and the solid dosage form behaves like oral solution. Currently biowaivers are determined based on solubility, permeability and dissolution, but the factors related to the gastrointestinal tract and the dynamic nature of drug dissolution and systemic pharmacokinetics are not taken into account. We utilized pharmacokinetic simulation model to study effects of formulation types, and different rates of dissolution and gastric emptying on drug concentrations in plasma. Simulated maximum concentration in plasma (C(max)) and area under the curve (AUC) values of solid dosage forms were compared to the simulations of oral solution. Based on simulations about half of BCS I drugs have higher risk to fail in bioequivalency (BE) study than BCS III drugs. For these BCS I compounds 10-25% differences of C(max) were observed. Rest of the BCS I drugs and all BCS III drugs have lower risk to fail in BE study since less than 10% difference in C(max) and AUC were observed. Pharmacokinetic simulation model was valuable tool to evaluate biowaiver criteria and to study the effects of drug and physiology gastrointestinal related factors on C(max) and AUC.

Level A in vitro-in vivo correlation (IVIVC) model with Bayesian approach to formulation series.

In vitro-in vivo correlation (IVIVC) models for formulation series are useful in drug development, but the current models are limited by their inability to include data variability in the predictions. Our goal was to develop a level A IVIVC model that provides predictions with probabilities. The Bayesian approach was used to describe uncertainty related to the model and the data. Three bioavailability studies of levosimendan were used to develop IVIVC model. Dissolution was tested at pH 5.8 with basket. The IVIVC model with Bayesian approach consisted of prior and observed data. All observed data were fitted to the one-compartment model together with prior data. Probability distributions of pharmacokinetic parameters and concentration time profiles were obtained. To test the external predictability of IVIVC model, only dissolution data of formulations E and F were used. The external predictability was good. The possibility to utilize all observed data when constructing IVIVC model, can be considered as a major strength of Bayesian approach. For levosimendan capsule data traditional IVIVC model was not predictable. The usefulness of IVIVC model with Bayesian approach was shown with our data, but the same approach can be used more widely for formulation optimization and for dissolution based biowaivers.

Biowaiver monographs for immediate release solid oral dosage forms: ranitidine hydrochloride.

Literature and experimental data relevant to the decision to allow a waiver of in vivo bioequivalence testing for the approval of immediate release (IR) solid oral dosage forms containing ranitidine hydrochloride are reviewed. According to the current Biopharmaceutics Classification System (BCS), ranitidine hydrochloride should be assigned to Class III. However, based on its therapeutic and therapeutic index, pharmacokinetic properties and data related to the possibility of excipient interactions, a biowaiver can be recommended for IR solid oral dosage forms that are rapidly dissolving and contain only those excipients as reported in this study.

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.