Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to zwitterionic ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP.

Analytical profiling and stability evaluation of liposomal drug delivery systems: A rapid UHPLC-CAD-based approach for phospholipids in research and quality control.

Phospholipids and their derivatives represent a broad range of multifunctional substances used as excipients or active ingredients by different industries due to their natural origin and unique properties. A fast and reliable quantification as well as comprehensive stability evaluation are of major importance in the process of development and quality control of lipid-based systems. Therefore, the present study is focused on the development and validation of a rapid ultra-high performance liquid chromatography - charged aerosol detector based (UHPLC-CAD) method for simultaneous detection of a multitude of natural and synthetic lipids, (charged) phospholipids, lipophilic fluorescent markers and their possible degradation products. Twenty-two compounds were characterized by a strong linear response of the detector (R2 > 0.97). Moreover, remarkable limits of detection (≤10 µg mL-1) and limits of quantification (≤25 µg mL-1) associated with a consistent reproducibility were achieved for all tested molecules. The performance of the analytical method was demonstrated by analyzing the lipid composition (after different production stages and photodegradation) of both bupivacaine loaded liposomes and a Doxil®-like formulation. The newly developed method combines a rapid, comprehensive, and efficient quantification with minimal economic effort and ecologic consequences, meeting the requirements of modern analytical processes and offering a broad range of possible applications in various industrial sectors and scientific laboratories.

Injectable Lipid-Based Depot Formulations: Where Do We Stand?

The remarkable number of new molecular entities approved per year as parenteral drugs, such as biologics and complex active pharmaceutical ingredients, calls for innovative and tunable drug delivery systems. Besides making these classes of drugs available in the body, injectable depot formulations offer the unique advantage in the parenteral world of reducing the number of required injections, thus increasing effectiveness as well as patient compliance. To date, a plethora of excipients has been proposed to formulate depot systems, and among those, lipids stand out due to their unique biocompatibility properties and safety profile. Looking at the several long-acting drug delivery systems based on lipids designed so far, a legitimate question may arise: How far away are we from an ideal depot formulation? Here, we review sustained release lipid-based platforms developed in the last 5 years, namely oil-based solutions, liposomal systems, in situ forming systems, solid particles, and implants, and we critically discuss the requirements for an ideal depot formulation with respect to the used excipients, biocompatibility, and the challenges presented by the manufacturing process. Finally, we delve into lights and shadows originating from the current setups of in vitro release assays developed with the aim of assessing the translational potential of depot injectables.

Delivery of Rapamycin Using In Situ Forming Implants Promotes Immunoregulation and Vascularized Composite Allograft Survival.

Vascularized composite allotransplantation (VCA), such as hand and face transplantation, is emerging as a potential solution in patients that suffered severe injuries. However, adverse effects of chronic high-dose immunosuppression regimens strongly limit the access to these procedures. In this study, we developed an in situ forming implant (ISFI) loaded with rapamycin to promote VCA acceptance. We hypothesized that the sustained delivery of low-dose rapamycin in proximity to the graft may promote graft survival and induce an immunoregulatory microenvironment, boosting the expansion of T regulatory cells (Treg). In vitro and in vivo analysis of rapamycin-loaded ISFI (Rapa-ISFI) showed sustained drug release with subtherapeutic systemic levels and persistent tissue levels. A single injection of Rapa-ISFI in the groin on the same side as a transplanted limb significantly prolonged VCA survival. Moreover, treatment with Rapa-ISFI increased the levels of multilineage mixed chimerism and the frequency of Treg both in the circulation and VCA-skin. Our study shows that Rapa-ISFI therapy represents a promising approach for minimizing immunosuppression, decreasing toxicity and increasing patient compliance. Importantly, the use of such a delivery system may favor the reprogramming of allogeneic responses towards a regulatory function in VCA and, potentially, in other transplants and inflammatory conditions.

Immobilization of plasmids in bacterial nanocellulose as gene activated matrix.

The synergy of the local delivery of nucleic acids using a hydrogel-based gene activated matrix (GAM) might support regenerative processes on a genetic level by concurrently providing a cell-friendly microenvironment. To investigate bacterial nanocellulose (BNC) as GAM, two plasmids (pSV-ß-Gal and pGL3) were incorporated by reswelling and injection techniques forming matrix and core-shell systems as determined by SEM and staining experiments. The release was found to be dependent on the type of BNC, the plasmid and the loading technique, and lasted over at least 20 days. No morphological or mechanical changes of the BNC due to the presence of plasmids were observed. Immobilized plasmids especially in the matrix systems were protected against enzymatic degradation by maintaining the high biocompatibility of BNC and transfection efficacy of the plasmids. These results indicate that BNC can be used as a promising and renewable carrier for the application as local gene delivery system.

Study on the in situ aggregation of liposomes with negatively charged phospholipids for use as injectable depot formulation.

Compared to conventional parenteral formulations injectable depot formulations, owing to a sustained drug release, offer several advantages, such as a reduced dosing frequency - and consequent improved compliance - or a predictable release profile. Additionally, fluctuations in the drug blood level may be smoothened and consequently side effects reduced. Because of their capability to encapsulate water soluble drugs and their very low toxicity profile liposomes comprising phospholipids, most certainly represent a vehicle of choice for subcutaneous (s.c.) or intramuscular (i.m.) administration typical for depot injections too. In the past, especially liposomes containing negatively charged phosphatidylserines were investigated regarding their aggregation and fusion behavior upon addition of calcium ions. Liposomes need to have a large size to prevent fast removal from the s.c. or i.m. injection site to make them useful as depot vehicle. In order to obtain such large liposomes, aggregation of smaller liposomes may be considered. Aim of the present study was to induce and investigate controlled aggregation of vesicles containing other negatively charged phospholipids besides phosphatidylserines upon mixing with a solution of divalent cations. L-α-phosphatidylcholine from egg (EPC) liposomes formulated with 25 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG) proved to be possible lipid-based depot candidates due to their strong aggregation induced by calcium and magnesium cations. Different aggregation profiles with both cations could be observed. Morphological investigations of the aggregates showed that individual liposomes remain stable in the aggregates and no fusion occurred. A fluorescence-based fusion assay confirmed these results. Differential scanning calorimetry measurements supported the findings of the diverse aggregation profiles with calcium or magnesium owing to different binding sites of the cations to the lipid molecules.