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The use of liposomes in improving diagnostic assays for a broad spectrum of medical, environmental, and civil defense purposes, is demonstrated in many scientific papers as described in detail elsewhere. The main advantage of the liposome-based diagnostic assay is that it combines the lack of need to use radioactivity with increased sensitivity due to spontaneous amplification. One aspect which may offset the increase in sensitivity due to use of liposomes is a reduction in the signal-to-noise ratio. The principle of the assay is to use the fluorescent sterically stabilized immunoliposomes having the specific MAb attached to their surface as a means to detect the presence of antigen recognized by the specific MAb. One of the major factors which may reduce the sensitivity of liposome-based fluorescence immunoassay is aggregation of the liposomes which may be induced by the medium components other than the analytes.

PURPOSE. Developing and testing of microbicides for pre-exposure prophylaxis and post-exposure protection from HIV are on the list of major HIV/AIDS research priorities. To improve solubility and bioavailability of highly potent anti-retroviral drugs, we explored the use of a nanoparticle (NP) for formulating a combination of two water-insoluble HIV inhibitors. METHODS. The combination of a non-nucleoside HIV reverse transcriptase inhibitor (NNRTI), Efavirenz (EFV), and an inhibitor of HIV integrase, Elvitegravir (ELV) was stabilized with a graft copolymer of methoxypolyethylene glycol-polylysine with a hydrophobic core (HC) composed of fatty acids (HC-PGC). Formulations were tested in TZM-bl cells infected either with wild-type HIV-1(IIIB), or drug-resistant HIV-1 strains. In vivo testing of double-labeled NP formulations was performed in female rats after a topical intravaginal administration using SPECT/CT imaging and fluorescence microscopy. RESULTS. We observed a formation of stable 23–30 nm NP with very low cytotoxicity when EFV and ELV were combined with HC-PGC at a 1:10 weight ratio. For NP containing ELV and EFV (at 1:1 by weight) we observed a remarkable improvement of EC50 of EFV by 20 times in the case of A17 strain. In vivo imaging and biodistribution showed in vivo presence of NP components at 24 and 48h after administration, respectively. CONCLUSIONS. Insoluble orthogonal inhibitors of HIV-1 life cycle may be formulated into the non-aggregating ultrasmall NP which are highly efficient against NNRTI-resistant HIV-1 variant.

Our objective was to develop novel nanocarriers (protected graft copolymer, PGC) that improve the stability of heparin binding EGF (HBEGF) and gastrin and then to use PGC-formulated HBEGF (PGC-HBEGF) and Omeprazole (+/- PGC-gastrin) for normalizing fasting blood glucose (FBG) and improving islet function in diabetic mice.HBEGF, PGC-HBEGF, Omeprazole, Omeprazole + PGC-HBEGF, Omeprazole + PGC-gastrin + PGC-HBEGF and epidermal growth factor (EGF) + gastrin were tested in multiple low dose streptozotocin diabetic mice.Omeprazole + PGC-HBEGF normalized FBG and is better than EGF + gastrin at improving islet function and decreasing insulitis. Groups treated with Omeprazole, Omeprazole + PGC-HBEGF, or EGF + gastrin have significantly improved islet function versus saline control. All animals that received PGC-HBEGF had significantly reduced islet insulitis versus saline control. Non-FBG was lower for Omeprazole + PGC-gastrin + PGC-HBEGF but Omeprazole + PGC-HBEGF alone showed better FBG and glucose tolerance.Omeprazole + PGC-HBEGF provides a sustained exposure to both EGFRA and gastrin, improves islet function, and decreases insulitis in multiple low dose streptozotocin diabetic mice. Although HBEGF or EGF elevates non-FBG, it facilitates a reduction of insulitis and, in the presence of Omeprazole, provides normalization of FBG at the end of treatment. The study demonstrates Omeprazole and PGC-HBEGF is a viable treatment for diabetes.

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150–250 nm stable nanoparticles (NP). The NP and HC-PGC were non-toxic in experiments with TZM-bl reporter cells. Nanoparticles containing selected efficient candidate Z107 NNRTI preserved the ability to inhibit HIV-1 reverse transcriptase polymerase activities with no appreciable change of EC50. The formulation with HC-PGC bearing residues of oleic acid resulted in nanoparticles that were nearly identical in anti-HIV-1 potency when compared to Z107 solutions in DMSO (EC50 = 7.5 ± 3.8 vs. 8.2 ± 5.1 nM). Therefore, hydrophobic core macromolecular stabilizers form nanoparticles with insoluble NNRTI while preserving the antiviral activity of the drug cargo.

Initially developed in 1992 as an MR imaging agent, the family of protected graft copolymers (PGC) is based on a conjugate of polylysine backbone to which methoxypoly(ethylene glycol) (MPEG) chains are covalently linked in a random fasion via N-ε-amino groups. While PGC is relatively simple in terms of its chemcial composition and structure, it has proved to be a versatile platform for in vivo drug delivery. The advantages of poly amino acid backbone grafting include multiple available linking sites for drug and adaptor molecules. The grafting of PEG chains to PGC does not compromise biodegradability and does not result in measurable toxicity or immunogenicity. In fact, the biocompatablility of PGC has resulted in its being one of the few 100% synthetic non-proteinaceous macromolecules that has suceeded in passing the initial safety phase of clinical trials. PGC is capable of long circulation times after injection into the blood stream and as such found use early on as a carrier system for delivery of paramagnetic imaging compounds for angiography. Other PGC types were later developed for use in nuclear medicine and optical imaging applications in vivo. Recent developments in PGC-based drug carrier formulations include the use of zinc as a bridge between the PGC carrier and zinc-binding proteins and re-engineering of the PGC carrier as a covalent amphiphile that is capabe of binding to hydrophobic residues of small proteins and peptides. At present, PGC-based formulations have been developed and tested in various disease models for: 1) MR imaging local blood circulation in stroke, cancer and diabetes; 2) MR and nuclear imaging of blood volume and vascular permeability in inflammation; 3) optical imaging of proteolytic activity in cancer and inflammation; 4) delivery of platinum(II) compounds for treating cancer; 5) delivery of small proteins and peptides for treating diabetes, obesity and myocardial infarction. This review summarizes the experience accumulated by various research groups that chose to use PGC as a drug delivery platform.

To develop a long-acting formulation of native human insulin with a similar pharmacodynamics (PD) profile as the insulin analogue insulin glargine (Lantus®, Sanofi-Aventis) with the expectation of retaining native human insulin’s superior safety profile as insulin glargine is able to activate the insulin-like growth factor 1 (IGF-1) receptor and is linked to a number of malignancies at a higher rate than regular human insulin. Development of protected graft copolymer (PGC) excipients that bind native human insulin non-covalently and testing blood glucose control obtained with these formulations in streptozotocin-induced diabetic Sprague Dawley rats compared to equally dosed insulin glargine. PGC-formulations of native human insulin are able to control blood glucose to the same extent and for the same amount of time after s.c. injection as the insulin analogue insulin glargine. No biochemical changes were made to the insulin that would change receptor binding and activation with their possible negative effects on the safety of the insulin. Formulation with the PGC excipient offers a viable alternative to biochemically changing insulin or other receptor binding peptides to improve PD properties.

Purpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy.

Purpose. To compare the performance of sterically stabilized, doxorubicin-loaded liposomes with and without surface attached specific antibodies (D-SSIL and D-SSL, respectively). Methods. Small (≤ 120 nm) unilamellar liposomes were prepared composed of hydrogenated soy phosphatidylcholine, hydrogenated phosphatidylethanolamine (HPE), cholesterol, and 2000Da polyethylene glycol (2000PEG) attached to the primary amino group of distearoyl phosphatidylethanolamine. Doxorubicin was remote-loaded into these liposomes by an ammonium sulfate gradient to form the D-SSL. Monoclonal IgG3 NI32/2 antibodies directed against a polyoma virus tumor-associated antigen expressed on A9 etc 102 murine fibrosarcoma cells were attached to the D-SSL HPE via a thioether bond to form the D-SSIL-32/2. A control of nonspecific D-SSIL was prepared by attaching nonspecific IgG3-enriched immunoglobulins to D-SSL. All liposomes were physically and chemically characterized and then tested in vitro for tumor cell binding, specificity, and uptake by macrophages; and in vivo for the drug plasma pharmacokinetics after intravenous administration in mice. Results. (i) The attachment of antibodies to D-SSL did not impair their chemical or physical stability and had a minimal effect on their size and level of loaded drug, (ii) The combination of specific antibodies and 2000PEG grafted in the liposomes improved the specific binding to relevant target cells by reducing the level of unspecific binding to nonrelevant cells. (iii) D-SSIL retained the prolonged circulation and slow clearance typical of SSL lacking the antibodies. Conclusions. Sterically stabilized immunoliposomes exhibited stability, ability to recognize target cells, and prolonged circulation time. This study also shows that it is feasible to prepare them in pharmaceutically acceptable dosage form. Thus, further investigation for tumor targeting and efficacy is warranted.

This short paper describes the problems, and their solutions, of formulating liposomal dosage forms for clinical use, as exemplified in tumor therapy by liposomal doxorubicin. We will demonstrate that the development of an efficacious drug delivery system requires fitting the delivery system to the micro-anatomy of the tumor. Many tumors have gaps in their capillarics which permit particles smaller than 120 nm to slowly extravasate into the tumor. Therefore small liposomes having a prolonged circulation time may be suitable drug carriers for tumor therapy. Recently a liposomal doxorubicin formulation (DOX-SL™) was recommended for approval by the US FDA, with indications of treating Kaposi's sarcoma. These liposomes seem to be efficacious in other types of tumors as well. The unique features of this formulation include: control of the biodistribution and pharmacokinetics of the intact liposomes through liposome lipid composition, especially the role of steric stabilization of the liposomes which e...

Purpose. To evaluate benefits in tumor localization, availability, and noncancerous organ distribution of doxorubicin (DOX) delivered via small (≤120 nm) sterically stabilized immunoliposomes targeted against a tumor-associated antigen in fibrosarcoma-bearing mice.

To determine and compare pharmacokinetics and toxicity of two nanoformulations of Vasoactive Intestinal Peptide (VIP). VIP was formulated using a micellar (Sterically Stabilized Micelles, SSM) and a polymer-based (Protected Graft Copolymer, PGC) nanocarrier at various loading percentages. VIP binding to the nanocarriers, pharmacokinetics, blood pressure, blood chemistry, and acute maximum tolerated dose (MTD) of the formulations after injection into BALB/c mice were determined. Both formulations significantly extend in vivo residence time compared to unformulated VIP. Formulation toxicity is dependent on loading percentage, showing major differences between the two carrier types. Both formulations increase in vivo potency of unformulated VIP and show acute MTDs at least 140 times lower than unformulated VIP, but still at least 100 times higher than the anticipated highest human dose, 1–5 μg/kg. These nanocarriers prevented a significant drop in arterial blood pressure compared to unformulated VIP. While both carriers enhance in vivo residence time compared to unformulated VIP and reduce the drop in blood pressure immediately after injection, PGC is the excipient of choice to extend residence time and improve the safety of potent therapeutic peptides such as VIP.

The amphipathic anthracycline base doxorubicin (DXR) was accumulated in the aqueous phase of the liposomes where it reached a level as high as 100-fold its concentration in the remote loading mediu...

OBJECTIVE— The pathogenesis of type 1 diabetes involves autoimmune lymphocytic destruction of insulin-producing β-cells and metabolic dysregulation. An early biomarker of pancreatic islet damage is islet microvascular dysfunction, and alterations in vascular volume, flow, and permeability have been reported in numerous models of type 1 diabetes. Consequently, the ability to noninvasively monitor the dynamics of the pancreatic microvasculature would aid in early diagnosis and permit the assessment, design, and optimization of individualized therapeutic intervention strategies. RESEARCH DESIGN AND METHODS— Here, we used the long circulating paramagnetic contrast agent, protected graft copolymer (PGC) covalently linked to gadolinium-diethylenetriaminepentaacetic acid residues (GdDTPAs) labeled with fluorescein isothiocyanate (PGC-GdDTPA-F), for the noninvasive semiquantitative evaluation of vascular changes in a streptozotocin (STZ)-induced mouse model of type 1 diabetes. Diabetic animals and nondiabetic controls were monitored by magnetic resonance imaging (MRI) after injection of PGC-GdDTPA-F. RESULTS— Our findings demonstrated a significantly greater accumulation of PGC-GdDTPA-F in the pancreata of diabetic animals compared with controls. MRI permitted the in vivo semiquantitative assessment and direct visualization of the differential distribution of PGC-GdDTPA-F in diabetic and control pancreata. Ex vivo histology revealed extensive distribution of PGC-GdDTPA-F within the vascular compartment of the pancreas, as well as considerable leakage of the probe into the islet interstitium. By contrast, in nondiabetic controls, PGC-GdDTPA-F was largely restricted to the pancreatic vasculature at the islet periphery. CONCLUSIONS— Based on these observations, we conclude that in the STZ model of type 1 diabetes, changes in vascular volume and permeability associated with early stages of the disease can be monitored noninvasively and semiquantitatively by MRI.

Background Currently available local anesthetics have relatively brief durations of action. An ultralong-acting local anesthetic would benefit patients with acute and chronic pain. The authors prepared and characterized a novel liposomal bupivacaine formulation using remote loading of bupivacaine along an ammonium sulfate gradient and assessed its efficacy in humans. Methods A large multivesicular liposomal bupivacaine formulation was prepared by subjecting small unilamellar vesicles to successive freeze-and-thaw cycles. Bupivacaine hydrochloride was then remotely loaded into the liposomes along an ammonium sulfate gradient ([(NH4)2SO4)]intraliposome/[(NH4)2SO4)]medium > 1000). The liposomes were then characterized for size distribution; drug-to-phospholipid ratio; in vitro release profile at 4 degree, 21 degree C, and 37 degree C; sterility; and pyrogenicity. Six subjects each received six intradermal injections in the lower back with 0.5 ml of 0.5, 1.0, and 2% liposomal bupivacaine; 0.5% standard bupivacaine; saline; and "empty" liposomes. Duration of analgesia was assessed using pinprick testing of the skin directly over the injection sites. Results were compared using the log-rank test. Results The mean large multivesicular vesicle size was 2439 +/- 544 nm, with a drug-to-phospholipid ratio of 1.8, fivefold greater than results previously reported. In vitro release was slowest at 4 degree C. The median duration of analgesia with 0.5% standard bupivacaine was 1 h. The median durations of analgesia after 0.5, 1.0, and 2.0% liposomal bupivacaine were 19, 38, and 48 h, respectively. Neither saline nor "empty" liposomes produced analgesia. Conclusions This novel liposomal formulation had a favorable drug-to-phospholipid ratio and prolonged the duration of bupivacaine analgesia in a dose-dependent manner. If these results in healthy volunteers can be duplicated in the clinical setting, this formulation has the potential to significantly impact the management of pain.

Formulation of peptides in a nanocarrier made up of protected-graft-copolymer (PGC) can greatly enhance pharmacokinetics and biodistribution of the peptides in vivo. Glucagon-like peptide 1 (GLP-1) is β-cell protective and decreases the severity symptoms of diabetes in Type 2 and streptozotocin models. We hypothesized that PGC-formulated GLP-1 can prevent Type 1 diabetes in NOD mice. 4-5 week old female NOD mice [n=25] were treated for eight weeks with 3mg/kg PGC-formulated GLP-1 administered s.c. either once or three times per week. After the treatment period, the mice were monitored for signs of diabetes until they reached 36 weeks of age. A group of NOD mice treated with anti-CD3 antibodies (0.5mg/kg i.p. once at treatment week 0 and once at treatment week 1) was used as a positive control for diabetes prevention. At 36 weeks of age, 48% of the mice treated with saline had developed diabetes. Anti-CD3 treatment prevented 50% of the diabetes cases in the mice with only 24% of the mice in this group having developed diabetes at 36 weeks of age. Once a week GLP-1 treatment (3mg/kg s.c.) was as effective in preventing diabetes as anti-CD3 treatment with only 28% of mice being diabetic at 36 weeks of age. Surprisingly, there was no difference between the three times per week GLP-1 treatment group (3mg/kg s.c.) and the saline control: 54.1% of the mice treated three times per week became diabetic, which was not statistically significantly different form the 48% diabetes incidence in the saline group.