Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNA in Mice.

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

DNA lipid nanoparticle vaccine targeting outer surface protein C affords protection against homologous Borrelia burgdorferi needle challenge in mice.

Introduction: The incidence of Lyme disease (LD) in Canada and the United States has risen over the last decade, nearing 480,000 cases each year. Borrelia burgdorferi sensu lato, the causative agent of LD, is transmitted to humans through the bite of an infected tick, resulting in flu-like symptoms and often a characteristic bull's-eye rash. In more severe cases, disseminated bacterial infection can cause arthritis, carditis and neurological impairments. Currently, no vaccine is available for the prevention of LD in humans. Methods: In this study, we developed a lipid nanoparticle (LNP)-encapsulated DNA vaccine encoding outer surface protein C type A (OspC-type A) of B. burgdorferi. Results: Vaccination of C3H/HeN mice with two doses of the candidate vaccine induced significant OspC-type A-specific antibody titres and borreliacidal activity. Analysis of the bacterial burden following needle challenge with B. burgdorferi (OspC-type A) revealed that the candidate vaccine afforded effective protection against homologous infection across a range of susceptible tissues. Notably, vaccinated mice were protected against carditis and lymphadenopathy associated with Lyme borreliosis. Discussion: Overall, the results of this study provide support for the use of a DNA-LNP platform for the development of LD vaccines.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Influence of bound dodecanoic acid on the reconstitution of albumin nanoparticles from a lyophilized state.

The development of reference standards for nanoparticle sizing allows for cross laboratory studies and effective transfer of particle sizing methodology. To facilitate this, these reference standards must be stable upon long-term storage. Here, we examine factors that influence the properties of cross-linked albumin nanoparticles, fabricated with an ethanol desolvation method, when reconstituted from a lyophilized state. We demonstrate, with nanoparticle tracking analysis, no significant changes in mean particle diameter upon reconstitution of albumin nanoparticles fabricated with bovine serum albumin loaded with dodecanoic acid, when compared to nanoparticles fabricated with a fatty acid-free BSA. We attribute this stability to the modulation of nanoparticle charge-charge interactions at dodecanoic acid specific binding locations. Furthermore, we demonstrate this in a lyophilized state over six months when stored at - 80 °C. We also show that the reconstitution process is readily transferable between technicians and laboratories and further confirm our finding with dynamic light scattering analysis.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.

BACKGROUND: Extracellular vesicles (EVs) produced by human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are currently investigated for their clinical effectiveness towards immune-mediated diseases. The large amounts of stem cell-derived EVs required for clinical testing suggest that bioreactor production systems may be a more amenable alternative than conventional EV production methods for manufacturing products for therapeutic use in humans. METHODS: To characterize the potential utility of these systems, EVs from four hBM-MSC donors were produced independently using a hollow-fiber bioreactor system under a cGMP-compliant procedure. EVs were harvested and characterized for size, concentration, immunophenotype, and glycan profile at three separate intervals throughout a 25-day period. RESULTS: Bioreactor-inoculated hBM-MSCs maintained high viability and retained their trilineage mesoderm differentiation capability while still expressing MSC-associated markers upon retrieval. EVs collected from the four hBM-MSC donors showed consistency in size and concentration in addition to presenting a consistent surface glycan profile. EV surface immunophenotypic analyses revealed a consistent low immunogenicity profile in addition to the presence of immuno-regulatory CD40 antigen. EV cargo analysis for biomarkers of immune regulation showed a high abundance of immuno-regulatory and angiogenic factors VEGF-A and IL-8. CONCLUSIONS: Significantly, EVs from hBM-MSCs with immuno-regulatory constituents were generated in a large-scale system over a long production period and could be frequently harvested with the same quality and quantity, which will circumvent the challenge for clinical application.

DNA Based Vaccine Expressing SARS-CoV-2 Spike-CD40L Fusion Protein Confers Protection Against Challenge in a Syrian Hamster Model.

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile.

Effects of Excipients on the Structure and Dynamics of Filgrastim Monitored by Thermal Unfolding Studies by CD and NMR Spectroscopy.

Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 µM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T 1, T 2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

Nanoparticle size and production efficiency are affected by the presence of fatty acids during albumin nanoparticle fabrication.

We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.

Evaluation of Microflow Digital Imaging Particle Analysis for Sub-Visible Particles Formulated with an Opaque Vaccine Adjuvant.

Microflow digital imaging (MDI) has become a widely accepted method for assessing sub-visible particles in pharmaceutical formulations however, to date; no data have been presented on the utility of this methodology when formulations include opaque vaccine adjuvants. This study evaluates the ability of MDI to assess sub-visible particles under these conditions. A Fluid Imaging Technologies Inc. FlowCAM® instrument was used to assess a number of sub-visible particle types in solution with increasing concentrations of AddaVax™, a nanoscale squalene-based adjuvant. With the objective (10X) used and the limitations of the sensor resolution, the instrument was incapable of distinguishing between sub-visible particles and AddaVax™ droplets at particle sizes less than 5 µm. The instrument was capable of imaging all particle types assessed (polystyrene beads, borosilicate glass, cellulose, polyethylene protein aggregate mimics, and lysozyme protein aggregates) at sizes greater than 5 µm in concentrations of AddaVax™ up to 50% (vol:vol). Reduced edge gradients and a decrease in measured particle sizes were noted as adjuvant concentrations increased. No significant changes in particle counts were observed for polystyrene particle standards and lysozyme protein aggregates, however significant reductions in particle counts were observed for borosilicate (80% of original) and cellulose (92% of original) particles. This reduction in particle counts may be due to the opaque adjuvant masking translucent particles present in borosilicate and cellulose samples. Although the results suggest that the utility of MDI for assessing sub-visible particles in high concentrations of adjuvant may be highly dependent on particle morphology, we believe that further investigation of this methodology to assess sub-visible particles in challenging formulations is warranted.

Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems.

Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209-290). Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)-Gen. Subj. 1830(12) (2013) 5515-5525; Kobayashi, Biologicals 34(1) (2006) 55-59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661-4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article 'Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa' where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.

Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin.

CONTEXT: At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein. OBJECTIVE: As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids. MATERIALS AND METHODS: Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry. RESULTS: Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7 d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed. DISCUSSION AND CONCLUSION: The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.

Investigation of the differences in thermal stability of two recombinant human serum albumins with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes by UV circular dichroism spectropolarimetry.

Previous studies have demonstrated that liposome-protein interactions can result in changes to the thermal stability of the protein. We utilized far-UV circular dichroism spectropolarimetry and fluorescence spectroscopy to investigate the interaction of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes with two recombinant human serum albumins (rHSA). We demonstrate that rHSA expressed in Oryza sativa (OsrHSA) has improved secondary structure thermal stability compared to rHSA expressed in Pichia pastoris (PprHSA). A similar stability profile was observed when comparing bovine serum albumin (BSA) and defatted bovine serum albumin (dfBSA), suggesting the presence of fatty acids may be responsible for the improved stability of OsrHSA. Addition of DPPC liposomes reduced the thermal stability of both OsrHSA and BSA, but not of PprHSA or dfBSA. DPPC liposomes may disrupt stabilizing native fatty acids on OsrHSA and BSA.

Identification of N- and C-terminal residues involved in MAP kinase phosphatase 3 (MKP3) interdomain binding and auto-inhibition.

Interdomain binding has been shown to play an important role in the regulation of MAP kinase phosphatase 3 (MKP3), a phosphatase involved in control of ERK signalling pathways. In this study the residues in N- and C-terminal domains responsible for MKP3 interdomain binding are identified. Peptides from the N-terminal substrate-binding domain of MKP3 were assessed for their ability to bind the C-terminal catalytic domain using surface plasmon resonance. The data indicate that the residues 77-97 (the Post-KIM peptide) in the MKP3 N-terminal domain are responsible for its binding to the C-terminal catalytic domain. Residues in the C-terminal domain that might be important to interdomain binding were identified using data in the existing literature. Variants in which these residues had been altered were examined by circular dichroism and enzymatic assays to ensure retention of their structure and catalytic properties before being assessed for their ability to bind the Post-KIM peptide. The data show that glutamic acid 248, asparagine 267 and, to a lesser extent, arginine 299 are important for the interaction between the MKP3 C-terminal and the N-terminal domains. The identified residues map to a region on the surface of the C-terminal domain that appears complementary to the N-terminal domain surface defined by the Post-KIM peptide. This interdomain binding site is distinct from the substrate interaction sites.

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry.

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN alpha-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN alpha-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN alpha-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN alpha-2b remains mostly unchanged at a variety of BSA to IFN alpha-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN alpha-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN alpha-2b secondary structure when BSA is in molar excess.

Influence of drug-to-lipid ratio on drug release properties and liposome integrity in liposomal doxorubicin formulations.

Recent studies have shown that the release properties of vincristine encapsulated in large unilamellar vesicles (LUV) can be regulated by varying the drug-to-lipid (D/L) ratio. In this work it is shown that the drug-to-lipid ratio technique for regulating drug release also applies to doxorubicin encapsulated in LUV. In particular it is shown that the half-times (T(1/2)) for doxorubicin release from distearoylphosphatidylcholine (DSPC)/cholesterol LUV in vitro can be increased more than six-fold by increasing the D/L ratio from 0.05 (wt/wt) to 0.39 (wt/wt). This behavior is consistent with the behavior expected for drugs that precipitate following accumulation into liposomes. It is shown that the release properties of ciprofloxacin--a drug that does not precipitate following accumulation into LUV--are not affected by the D/L ratio. It is also shown that the crystalline intravesicular doxorubicin precipitates observed as the D/L ratio is raised from 0.05 to 0.46 adopt increasingly unusual morphologies. Linear crystals are observed at lower D/L values, however triangular and rectangular variations are observed as the D/L ratio is increased, and induce considerable distortion in vesicle morphology. It is noted that trapping efficiency following uptake of external doxorubicin into LUV is reduced from nearly 100% at a D/L ratio of 0.05 (wt/wt) to less than 70% at an (initial) D/L ratio of 0.8 (wt/wt). It is suggested that this arises, at least in part, from membrane-disrupting effects of internal drug crystals as they increase in size.

Characterization of the drug retention and pharmacokinetic properties of liposomal nanoparticles containing dihydrosphingomyelin.

The drug retention and circulation lifetime properties of liposomal nanoparticles (LN) containing dihydrosphingomyelin (DHSM) have been investigated. It is shown that replacement of egg sphingomyelin (ESM) by DHSM in sphingomyelin/cholesterol (Chol) (55/45; mol/mol) LN results in substantially improved drug retention properties both in vitro and in vivo. In the case of liposomal formulations of vincristine, for example, the half-times for drug release (T(1/2)) were approximately 3-fold longer for DHSM/Chol LN as compared to ESM/Chol LN, both in vitro and in vivo. Further increases in T(1/2) could be achieved by increasing the drug-to-lipid ratio of the liposomal vincristine formulations. In addition, DHSM/Chol LN also exhibit improved circulation lifetimes in vivo as compared to ESM/Chol LN. For example, the half-time for LN clearance (Tc(1/2)) at a low lipid dose (15 micromol lipid/kg, corresponding to 8 mg lipid/kg body weight) in mice was 3.8 h for ESM/Chol LN compared to 6 h for DHSM/Chol LN. In addition, it is also shown that DHSM/Chol LN exhibit much longer half-times for vincristine release as compared to LN with the "Stealth" lipid composition. It is anticipated that DHSM/Chol LN will prove useful as drug delivery vehicles due to their excellent drug retention and circulation lifetime properties.

Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations.

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid (D/L) ratios exhibit reduced release rates. In this work, the effects of very high D/L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T(1/2)) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T(1/2) = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T(1/2) = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/L ratio increased from 0.025 to 0.1 (wt/wt) (T(1/2) = 6.1-15.6 h respectively) but decreased at higher D/L ratios (D/L = 0.6, wt/wt) (T(1/2) = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.