Extended Circulation Half-life Research Articles

Preserved structure and function of human serum albumin self-folded in the oxidative cytoplasm of Escherichia coli

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.

Abstract 5118: Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner

Abstract Glypican 2 (GPC2) chimeric antigen receptor (CAR) T cells are safe and efficacious in neuroblastoma and are currently being tested in a first-in-human Phase 1 clinical trial at the Children’s Hospital of Philadelphia. Tumor-derived extracellular vesicles (TEVs) are nanosized vesicles secreted by cancer cells, often enriched with glypicans. However, the presence of GPC2 on neuroblastoma TEVs and any interaction with GPC2 CAR T cells has not been explored. High-levels of GPC2 were found on TEVs isolated from neuroblastoma preclinical models [7 cell lines, circulating EVs from 6 patient-derived xenograft (PDX)-bearing mice] and from the peripheral blood of 16 neuroblastoma patients, but not on circulating EVs from 7 non-tumor bearing mice or 12 healthy human donors. The level of circulating GPC2+ EVs isolated from the peripheral blood of PDX-bearing mice positively correlated with PDX size (R=0.96; P<0.0001) and parent PDX GPC2 expression. To evaluate how GPC2 on EVs modulates CAR T cell functionality, we generated EVs with varied levels of GPC2 (Hi/Lo/Neg) and co-incubated each EV subset with GPC2 CAR T cells. Here using immunofluorescence and flow assays we found that EVs bind GPC2 CAR T cells proportional to the amount of GPC2 on their surface. Further, GPC2Hi EV-CAR T cell synapses induced potent T cell activation shown by CD69/Granzyme B expression and release of IL-2/IFN-γ. GPC2Hi EV pre-incubated CAR T cells also displayed enhanced target neuroblastoma cell cytotoxicity, inducing 4-fold more specific cytotoxicity than CAR T cells incubated with control GPC2Neg EVs. In vivo we observed that injection of GPC2Hi EVs into GPC2Lo neuroblastoma SK-N-AS xenografts genetically altered to have significantly decreased endogenous EV production (via RAB27A knock-out) greatly enhanced the efficacy of co-infused GPC2 CAR T cells (p<0.05). Similarly, in an isogenic SK-N-AS-GPC2 xenograft murine model treated with a limiting number of GPC2 CAR T cells concurrently with the EV-inhibiting drug GW4869, GPC2 CAR efficacy was dependent on the presence of GPC2Hi EVs. Here, pharmacologic EV inhibition in mice decreased GPC2 CAR efficacy which could be rescued by intratumoral injection of GPC2Hi EVs. In both in vivo models, the presence of intratumoral GPC2Hi EVs resulted in a significantly enhanced tumor infiltration of activated (CD69+/CD25+) GPC2 CAR T cells (p<0.05). Taken together, GPC2Hi EVs are selectively secreted from neuroblastomas and bind and activate GPC2 CAR T cells enhancing their anti-tumor cytotoxicity. To further capitalize on these findings, we engineered GPC2Hi EVs to have an extended circulation half-life through albumin binding or tumor targeting via GD2 binding that are currently being tested in vivo and results will be reported. EVs offer a versatile platform for CAR antigen presentation and should be further validated as a strategy to enhance CAR T cell efficacy for solid tumors. Citation Format: Anna M. Giudice, Stephanie Matlaga, Guillem Pascual-Pasto, Patrick M. Schuerch, Geoffrey Rouin, Brendan McIntyre, Vincent P. Zecchino, Kristopher R. Bosse. Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5118.

Advancing healthcare: Exploring recent innovations in drug delivery systems

It has been demonstrated that drug delivery systems can enhance therapeutic efficacy, decrease toxicity, increase patient compliance, and even enable whole new medical treatments. Drug delivery systems are technological processes that shape and preserve drug molecules into forms that are appropriate for administration, such as tablets or liquids. They expedite drug delivery to the precise targeted region in the body, maximising therapeutic effectiveness and reducing buildup of off-target substances in the body. Modern drug delivery systems (DDS) are designed with the aid of cutting-edge technology to accelerate systemic drug delivery to the intended target location, hence increasing therapeutic efficacy and reducing accumulation off-target in the body. As a result, they are crucial to the management and treatment of disease. These (DDS) are multifunctional, biocompatible, biodegradable, and have high viscoelasticity with an extended circulation half-life. They are made of nanomaterials or miniature electronics. This review covers the advanced information regarding Drug Delivery Systems and critical analysis of its importance to enhance drug efficacy.

Calcium-mineralized polypeptide nanoparticle for intracellular drug delivery in osteosarcoma chemotherapy

The acidic microenvironments of tumor tissue and cells provide an opportunity for the development of pH-responsive drug delivery systems in cancer therapy. In this work, we designed a calcium carbonate (CaCO3)-core-crosslinked nanoparticle of methoxy poly(ethylene glycol)-block-poly(l-glutamic acid) through mineralization for intracellular delivery of doxorubicin (DOX), referred to as CaNP/DOX. CaNP/DOX exhibited high drug loading capability, uniform nanoparticle size, and pH-dependent DOX release. In the meantime, the enhanced cell uptake, superior cytotoxicity toward mouse osteosarcoma K7 cells, extended circulation half-life, and improved accumulation of DOX in K7 allograft tumor from CaNP/DOX were also demonstrated. More interestingly, CaNP/DOX displayed improved antitumor effect and reduced side effects against the K7 osteosarcoma-allografted mouse model and the 143B orthotopic osteosarcoma mouse model. Given the superior properties of Ca-mineralized polypeptide nanoparticle for intracellular drug delivery, the smart drug delivery system showed strong competitiveness in clinical chemotherapy of cancers.

An Albumin Sandwich Enhances in Vivo Circulation and Stability of Metabolically Labile Peptides.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB)2, as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB)2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB)2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.

The pH Sensitive Atram Peptide Hitchhikes on Human Serum Albumin En Route to Target Diseased Acidic Tissues

Our lab developed the acidity triggered rational membrane (ATRAM) peptide to target the acidic extracellular environment of tumors. Biophysical and cellular experiments have shown that the membrane interaction of ATRAM is pH-dependent. Often rapid enzymatic degradation and renal clearance of peptides in vivo restricts their successful application as a drug delivery system. However, our previous mice studies demonstrated that ATRAM has an extended circulation half-life. We hypothesized that ATRAM might be interacting with a blood component that shields the peptide from degradation. Binding assays confirmed that ATRAM binds to human serum albumin (HSA), a plasma protein, with higher affinity than insulin detemir, a clinically used drug, suggesting that it might bind to this protein in the blood stream. Fluorescence studies established that ATRAM interacts reversibly with HSA, as the peptide partitioned to lipid membranes after pre-incubation with HSA. This result suggests that while that peptide can use albumin as a carrier in the blood stream, it will still target and transfer to the cell membrane. Understanding how ATRAM is able to avoid immediate degradation and renal clearance in vivo can broaden the design and application range of peptides as therapeutics.

Biomimetic surface modification of discoidal polymeric particles

The rationale for the design of drug delivery nanoparticles is traditionally based on co-solvent self-assembly following bottom-up approaches or in combination with top-down approaches leading to tailored physiochemical properties to regulate biological responses. However, the optimal design and control of material properties to achieve specific biological responses remain the central challenge in drug delivery research. Considering this goal, we herein designed discoidal polymeric particles (DPPs) whose surfaces are re-engineered with isolated red blood cell (RBC) membranes to tailor their pharmacokinetics. The RBC membrane-coated DPPs (RBC-DPPs) were found to be biocompatible in cell-based in vitro experiments and exhibited extended blood circulation half-life. They also demonstrated unique kinetics at later time points in a mouse model compared to that of bare DPPs. Our results suggested that the incorporation of biomimicry would enable the biomimetic particles to cooperate with systems in the body such as cells and biomolecules to achieve specific biomedical goals.

NRG1-Fc improves metabolic health via dual hepatic and central action.

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

In vivo efficacy, toxicity and biodistribution of ultra-long circulating desferrioxamine based polymeric iron chelator

Desferrioxamine (DFO) is currently in clinical use to remove iron from transfusion-dependent patients with β-thalassemia major, sickle-cell anemia and the myelodysplastic syndromes. However, its short half-life, burdensome, subcutaneous mode of administration and propensity to cause neurotoxicity at high doses greatly hinder its use. Thus, developing an optimized version of DFO with extended half-life, and reduced toxicity is a major goal. Using high molecular weight (MW), non-toxic, hyperbranched polyglycerol with high functionality, we demonstrate that the efficacy of DFO can be tuned with considerable reduction in toxicity. Using zebrafish embryos and mice, we tested toxicity, iron removal efficacy with low dosing and the biodistribution of ultra-long circulating DFO (ULC-DFO) conjugates. There was no significant difference in the mortality and development of zebrafish embryos upon exposure to ULC-DFO. Similarly, body weights and serum lactate dehydrogenase levels in mice treated with ULC-DFO remained within the normal range throughout the tolerance study. Moreover, ULC-DFO is significantly more effective than low MW DFO in promoting iron removal both from organs and via urine in iron overloaded mice despite using a moderate, once-weekly dosing schedule. This is probably due to the extended circulation half-life of ULC-DFO. The MW of ULC-DFO influences the accumulation and biodistribution, with highest MW (637KDa) associated with up to 12% accumulation in the liver. In contrast, ULC-DFO with MWs of 75KDa and lower were associated with relatively low organ accumulation, indicating that biodistribution of ULC-DFO can be tuned. Since ULC-DFO has improved iron removal properties, longer plasma retention time and possesses excellent biocompatibility, it represents a polymer conjugate with high clinical utility in comparison to DFO for the treatment of transfusion dependent iron overload. More importantly, ULC-DFO is anticipated to reduce the requirement for prolonged subcutaneous infusion of DFO.

Parenterally administrable nano-micelles of 3,4-difluorobenzylidene curcumin for treating pancreatic cancer

Pancreatic cancer remains one of the most devastating diseases in terms of patient mortality rates for which current treatment options are very limited. 3,4-Difluorobenzylidene curcumin (CDF) is a nontoxic analog of curcumin (CMN) developed in our laboratory, which exhibits extended circulation half-life, while maintaining high anticancer activity and improved pancreas specific accumulation in vivo, compared with CMN. CDF however has poor aqueous solubility and its dose escalation for systemic administration remains challenging. We have engineered self-assembling nano-micelles of amphiphilic styrene-maleic acid copolymer (SMA) with CDF by non-covalent hydrophobic interactions. The SMA-CDF nano-micelles were characterized for size, charge, drug loading, release, serum stability, and in vitro anticancer activity. The SMA-CDF nano-micelles exhibited tunable CDF loading from 5 to 15% with excellent aqueous solubility, stability, favorable hemocompatibility and sustained drug release characteristics. The outcome of cytotoxicity testing of SMA-CDF nano-micelles on MiaPaCa-2 and AsPC-1 pancreatic cancer cell lines revealed pronounced antitumor response due to efficient intracellular trafficking of the drug loaded nano-micelles. Additionally, the nano-micelles are administrable via the systemic route for future in vivo studies and clinical translation. The currently developed SMA based nano-micelles thus portend to be a versatile carrier for dose escalation and targeted delivery of CDF, with enhanced therapeutic margin and safety.

Progress and Problems in Nutraceuticals Delivery

Nutraceuticals have always been considered as natural and safe supplements that may prevent disease, may substitute prescription drugs, may compensate for a poor diet or promote health. Several nutraceuticals products are in the market at present most of which are antioxidants and probiotics. The majority of nutraceutical products are intended for oral administration however; concerns related to their formulation, bioavailability and/or their site specific delivery arise. The labile nature, oral absorption and target-ability are the major underlying causes for poor nutraceuticals oral delivery. Investigators attempted to circumvent these obstacles by wide spectrum approaches tailored for each specific nutraceutical with solubility-enhancement techniques being the most commonly tackled. Recently, nanosizing techniques are utilized to answer the problem of nutraceuticals delivery with promising results in terms of nutraceutical-protection and stability on shelf and in vivo, solubility and dissolution rate enhancement, intestinal permeability improvement, intracellular and subcellular organelle targeting as well as extended circulation half-life all of which result in boosted bioavailability. The present review elucidates the progress and problems in the oral delivery of nutraceuticals.

Preparation and Characterization of Albumin Conjugates of a Truncated Peptide YY Analogue for Half-Life Extension

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Nanoparticle Encapsulated L-Asparaginase

Amino acid depletion with catabolizing enzymes is a potential therapeutic modality for many cancer types. L-asparaginase has been used to treat Acute Lymphoblastic Leukemia (ALL) for many decades and is part of treatment regimen that has a roughly 80% cure rate. The current frontline formulation is L-asparaginase from E.coli that has been conjugated to polyethylene glycol (PEG) for extended circulation half life and protection from immune responses. However, neutralizing antibodies still occur in a subset of patients, necessitating a switch to a different L-asparaginase, such as the less active enzyme from Erwinia chrysanthemi. This complicates therapy, risks severe allergic reactions, and if antibodies develop to the second enzyme the options become limited. The threat of immune reactions, as well as other side effects, has largely restricted L-asparaginase therapy to ALL, despite in vitro evidence that many other cancers might be sensitive to asparagine depletion.Our group has developed a novel nanoparticle enzyme encapsulation technology that overcomes the limitation of immune responses to enzymes or PEG. These particles, termed Synthetic Hollow Enzyme Loaded Porous nanoShells (SHELS), are produced in a two-step process where the first creates silica shells with large pores through which the enzyme can be loaded, and a second sealing step that encapsulates the mesoporous shell and the loaded enzyme cargo with a nanoporous silica layer. The work below was done with 200nm particles that have a total shell wall thickness of approximately 10nm.SHELS were loaded with L-asparaginase (Elspar). No loss of activity was seen when enzyme activity was measured in vitro. Groups of mice were injected IM with 5 IU of either free or SHELS encapsulated L-asparaginase. In each case, half of the mice were passively immunized with rabbit polyclonal anti-asparaginase antibodies to a blood concentration of ∼0.5 mg/ml. Serum asparaginase levels pre and post injection were measured by HPLC at 2, 5 and 8 days. Both free and encapsulated enzyme produced complete depletion of asparagine at day 2, but by day 5 asparagine levels had returned to baseline in the animals given free enzyme, whereas those given encapsulated enzyme maintained depletion at day 5 with restoration to baseline at day 8. There was no asparagine depletion in immunized animals with the free L-asparaginase, but SHELS encapsulated asparaginase produced an identical depletion both in the presence or absence of the neutralizing antibodies, indicating that the SHELS effectively shield the enzyme from the antibodies. Experiments in tumor models are ongoing as well as dose response and repeated administration studies. Disclosures:No relevant conflicts of interest to declare.

Effects of interferon-gamma liposomes targeted to platelet-derived growth factor receptor–beta on hepatic fibrosis in rats

No drugs have been approved clinically for the therapy of hepatic fibrosis. Though interferon-γ (IFN-γ) is a highly effective anti-fibrotic agent in vitro and in some animal models in vivo, its anti-fibrotic potential in clinical trials has been disappointing, due to unwanted off-target effects and a short half-life period which results in poor efficacy. The aims of this study are to develop a new targeted drug delivery system to selectively deliver IFN-γ to hepatic stellate cells (HSCs) and to investigate whether it will improve the anti-fibrotic effect of IFN-γ and reduce its side effects in fibrotic livers. Sterically stable liposomes (SSLs) were modified by cyclic peptides (pPB) with a specific affinity for platelet-derived growth factor receptor-β (PDGFR-β), and then IFN-γ was encapsulated in the targeted liposomes (pPB-SSL-IFN-γ). In vitro, pPB-SSL was found to be taken up and internalized by cultured activated HSCs. The binding of FITC-labeled pPB-SSL to activated HSCs was in a time-dependent and concentration-dependent manner, which could be inhibited by excess unlabelled pPB-SSL, PDGF-BB, suramin or monensin. The inhibitory effect of pPB-SSL-IFN-γ on the proliferation of activated HSCs was respectively 7.24-fold and 2.95-fold higher than that of free IFN-γ and IFN-γ encapsulated in untargeted SSLs. In healthy rats, the tissue distribution, living-body tracing image analyses and pharmacokinetics study showed that pPB-SSL-IFN-γ accumulated mainly in the livers and had a longer half-life than free IFN-γ (3.98±0.52h vs. 0.21±0.03h). Furthermore, in rats with hepatic fibrosis induced by thioacetamide injection, FITC-labeled pPB-SSL was found to predominantly localize in activated HSCs by immunofluorescent double staining for FITC and albumin or α-smooth muscle actin (α-SMA). The enhanced anti-fibrotic effect of pPB-SSL-IFN-γ treatnment was indicated by significant decreases in the histologic Ishak stage, collagen I-staining positive areas, and α-SMA expression levels in fibrotic livers. In addition, pPB-SSL-IFN-γ treatment improved the leukopenia caused by low- and high-dosage free IFN-γ treatments. In conclusion, IFN-γ encapsulated in pPB-SSL had an extended circulation half-life and was selectively delivered to activated HSCs, which enhanced the anti-fibrotic effect of IFN-γ and reduced its side-effects in rats with hepatic fibrosis. Thus, pPB-SSL-IFN-γ may be an effective agent for the therapy of hepatic fibrosis.

Overcoming in vivo barriers to targeted nanodelivery

Nanoparticles have been investigated as promising nanocarriers for delivery of imaging and therapeutic agents for several decades, but have met with limited success. Although enormous progress in the fields of nanotechnology and nanoscience has been achieved, basic discoveries have not yet translated into effective targeted therapies. Nanoparticles can potentially improve the pharmacokinetics and pharmacodynamics of drugs; however, the complexity of in vivo systems imposes multiple barriers that severely inhibit efficiency and have to be overcome to fully exploit the theoretical potential of nanoparticles. Here, we address two major challenges to effective systemic nanodelivery. Both limited penetration across the vascular endothelium and uptake by the reticuloendothelial system (RES) substantially impede effectiveness of nanoparticle delivery into tissues. Although the design of nanoparticles with extended circulation half-life is essential, it is not sufficient for effective penetration of nanoparticles across the formidable barrier formed by the vascular endothelium. Current nanodelivery systems rely on passive transvascular exchange and tissue accumulation. They require high dosages to create large concentration gradients that drive nanoparticles passively across the blood-tissue interface. However, passive accumulation has resulted in only a fractional dosage of nanoparticles penetrating into target tissue. This inevitably diminishes therapeutic efficacy and aggravates potential side effects. Although there are multiple ways to augment passive delivery, active delivery of targeted nanoparticles across the vascular endothelium could significantly increase the therapeutic index and decrease side effects of nanoparticle-based drug delivery systems. Use of active transendothelial transport pathways, such as caveolae, may provide an effective solution to both target and deliver nanoparticles.

Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors

Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors.

99mTc-labeling of lipid vesicles containing the lipophilic chelator PE-DTTA: Effect of tin-to-chelate ratio, chelate content and surface polymer on labeling efficiency and biodistribution behavior

When injected intravenously, lipid vesicles labeled with 99mTc by means of a lipophilic chelator dipalmitoylphosphatidylethanolamine-diethylenetriaminetetraacetic acid (PE-DTTA) are rapidly accumulated by the mononuclear phagocytic system (MPS). By derivatizing the membrane surface with the lipid-polymer complex dipalmitoylphosphatidylethanolamine-monomethoxy polyethylene glycol 5000 (PE-MPEG), MPS uptake can be suppressed and loss of 99mTc label from the lipid surface reduced depending upon both PE-DTTA and PE-MPEG content. For vesicles containing 20% PE-DTTA, addition of PE-MPEG makes no difference to their rate of clearance from the circulation. However for vesicles containing 2% PE-DTTA, addition of more than 0.8% PE-MPEG increases circulation half-life, suppresses liver uptake and reduces renal clearance of the 99mTc label. The molar ratio of reducing agent (Sn) to chelator (PE-DTTA) is critical to efficient and reproducible labeling. For vesicles containing 2% PE-DTTA at a lipid concentration of 100 mM, a Sn/DTTA ratio of 0.35 gives close to optimal labeling. Variation in the Sn/DTTA ratio by a factor of two negatively impacts upon both labeling efficiency in vitro and circulation half-life in vivo. Potential uses for technetium-labeled lipid vesicles with extended circulation half-life are discussed.