Oligo(Lactic Acid)8-Rapamycin Prodrug-Loaded Poly(Ethylene Glycol)-block-Poly(Lactic Acid) Micelles for Injection.

PURPOSE: To prepare an oligo(lactic acid)8-rapamycin prodrug (o(LA)8-RAP)-loaded poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) micelle for injection and characterize its compatibility and performance versus a RAP-loaded PEG-b-PLA micelle for injection in vitro and in vivo. METHODS: Monodisperse o(LA)8 was coupled on RAP at the C-40 via DCC/DMAP chemistry, and conversion of o(LA)8-RAP prodrug into RAP was characterized in vitro. Physicochemical properties of o(LA)8-RAP- and RAP-loaded PEG-b-PLA micelles and their antitumor efficacies in a syngeneic 4 T1 breast tumor model were compared. RESULTS: Synthesis of o(LA)8-RAP prodrug was confirmed by 1H NMR and mass spectroscopy. The o(LA)8-RAP prodrug underwent conversion in PBS and rat plasma by backbiting and esterase-mediated cleavage, respectively. O(LA)8-RAP-loaded PEG-b-PLA micelles increased water solubility of RAP equivalent to 3.3 mg/ml with no signs of precipitation. Further, o(LA)8-RAP was released more slowly than RAP from PEG-b-PLA micelles. With added physical stability, o(LA)8-RAP-loaded PEG-b-PLA micelles significantly inhibited tumor growth relative to RAP-loaded PEG-b-PLA micelles in 4 T1 breast tumor-bearing mice without signs of acute toxicity. CONCLUSIONS: An o(LA)8-RAP-loaded PEG-b-PLA micelle for injection is more stable than a RAP-loaded PEG-b-PLA micelle for injection, and o(LA)8-RAP converts into RAP rapidly in rat plasma (t1/2 = 1 h), resulting in antitumor efficacy in a syngeneic 4 T1 breast tumor model.

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic.

PURPOSE: To develop PEGylated variants of pUR4/FUD (FUD), a fibronectin assembly inhibitor, using 10 kDa, 20 kDa, and 40 kDa PEGs to evaluate their binding affinity and inhibitory potency. METHODS: The FUD peptide was recombinantly expressed, purified, and PEGylated at the N-terminus using 10 kDa, 20 kDa, and 40 kDa methoxy-PEG aldehyde. The PEGylates were purified and fractionated using ion-exchange chromatography. The molecular weight and degree of PEGylation of each conjugate was verified using MALDI-TOF. The binding affinity of each PEG-FUD conjugate was studied using isothermal titration colorimetry (ITC) and their inhibitory potency was characterized by a cell-based matrix assembly in vitro assay. RESULTS: The 10 kDa, 20 kDa, and 40 kDa PEG-FUD conjugates were synthesized and isolated in good purity as determined by HPLC analysis. Their molecular weight was consistent with attachment of a single PEG molecule to one FUD peptide. The binding affinity (Kd) and the fibronectin fibrillogenesis inhibitory potency (IC50) of all PEG-FUD conjugates remained nanomolar and unaffected by the addition of PEG. CONCLUSIONS: Retention of FUD fibronectin binding activity following PEGylation with three different PEG sizes suggest that PEG-FUD holds promise as an effective anti-fibrotic with therapeutic potential and a candidate for further pharmacokinetic and biodistribution studies.

Antifungal Efficacy of an Intravenous Formulation Containing Monomeric Amphotericin B, 5-Fluorocytosine, and Saline for Sodium Supplementation.

PURPOSE: Amphotericin B (AmB) and 5-fluorocytosine (5-FC) exhibit additive to synergistic activity against systemic mycoses. Incompatibility of prescribed formulations precludes concomitant IV administration, a route with distinct advantages. Previously, we used PEG-DSPE micelles to produce a reformulation of Fungizone (AmB-SD), AmB solubilized by sodium deoxycholate, called mAmB-90. Herein, we describe a second reformulation that facilitates co-delivery of mAmB-90 and 5-FC, and evaluate the effect of PEG-DSPE micelles on the combination's activity against Candida albicans. METHODS: We assessed the effect of 5-FC addition on the stability, in vitro toxicity, and antifungal efficacy of mAmB-90. The aggregation state and particle size of mAmB-90 combined with 5-FC (FmAmB-90) was evaluated over 48 h. Hemolytic activity was measured in vitro. Antifungal activity was determined in vitro against C. albicans. The efficacy of monotherapy and combination treatment was evaluated in a neutropenic mouse model of disseminated candidiasis. RESULTS: The aggregation state, particle size, and hemolytic activity of mAmB-90 were unaffected by 5-FC. While antifungal activity was similar in vitro, mAmB-90 alone and combined with 5-FC was more potent than AmB-SD in vivo. CONCLUSIONS: Short-term stability and in vivo efficacy of our formulation suggest potential to simultaneously deliver AmB and 5-FC for potent antifungal efficacy.

Oligo(lactic acid)n-Paclitaxel Prodrugs for Poly(ethylene glycol)-block-poly(lactic acid) Micelles: Loading, Release, and Backbiting Conversion for Anticancer Activity.

Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles are nanocarriers for poorly water-soluble anticancer agents and have advanced paclitaxel (PTX) to humans due to drug solubilization, biocompatibility, and dose escalation. However, PEG-b-PLA micelles rapidly release PTX, resulting in widespread biodistribution and low tumor exposure. To improve delivery of PTX by PEG-b-PLA micelles, monodisperse oligo(l-lactic acid), o(LA)8 or o(LA)16, has been coupled onto PTX at the 7-OH position, forming ester prodrugs: o(LA)8-PTX and o(LA)16-PTX, respectively. As expected, o(LA)n-PTX was more compatible with PEG-b-PLA micelles than PTX, increasing drug loading from 11 to 54%. While in vitro release of PTX was rapid, resulting in precipitation, o(LA)n-PTX release was more gradual: t1/2 = 14 and 26 h for o(LA)8-PTX and o(LA)16-PTX, respectively. Notably, o(LA)8-PTX and o(LA)16-PTX in PEG-b-PLA micelles resisted backbiting chain end scission, based on reverse-phase HPLC analysis. By contrast, o(LA)8-PTX and o(LA)16-PTX degraded substantially in 1:1 acetonitrile:10 mM PBS, pH 7.4, at 37 °C, generating primarily o(LA)2-PTX. The IC50 value of o(LA)2-PTX was ∼2.3 nM for A549 human lung cancer cells, equipotent with PTX in vitro. After weekly IV injections at 20 mg/kg as PEG-b-PLA micelles, o(LA)8-PTX induced tumor regression in A549 tumor-bearing mice, whereas PTX delayed tumor growth. Surprisingly, o(LA)8-PTX caused less toxicity than PTX in terms of change in body weight. In conclusion, o(LA)n acts as a novel promoiety, undergoing backbiting conversion without a reliance on metabolizing enzymes, and o(LA)n-PTX improves PTX delivery by PEG-b-PLA micelles, providing a strong justification for clinical evaluation.

Reformulation of Fungizone by PEG-DSPE Micelles: Deaggregation and Detoxification of Amphotericin B.

PURPOSE: Fungizone® (AmB-SD), amphotericin B solubilized by sodium deoxycholate, contains a highly aggregated form of the antifungal agent that causes dose-limiting renal toxicity. With the aim of reducing the formulation's toxicity by co-delivering monomeric amphotericin B (AmB) and sodium supplementation, we deaggregated AmB-SD with FDA-approved excipient PEG-DSPE in 0.9% NaCl-USP. Herein, we describe a reformulated AmB-SD with PEG-DSPE micelles that results in a less toxic drug with maintained antifungal activity. METHODS: We compared the aggregation state and particle size of AmB-SD alone or combined with PEG-DSPE micelles. In vitro hemolytic activity and in vivo renal toxicity were measured to determine the toxicity of different formulations. In vitro antifungal assays were performed to determine differences in efficacy among formulations. RESULTS: PEG-DSPE micelles in saline deaggregated AmB-SD. Deaggregated AmB-SD exhibited significantly reduced in vitro and in vivo toxicity. In vitro antifungal studies showed no difference in minimum inhibitory and fungicidal concentrations of AmB-SD combined with PEG-DSPE relative to the drug alone. CONCLUSIONS: Reformulation of AmB-SD with PEG-DSPE micelles in saline facilitates co-delivery of monomeric AmB and sodium supplementation, potentially reducing the dose-limiting nephrotoxicity of AmB-SD. Ease of preparation and commercially available components lead us to acknowledge its potential for clinical use.

Proof-of-Concept of Polymeric Sol-Gels in Multi-Drug Delivery and Intraoperative Image-Guided Surgery for Peritoneal Ovarian Cancer.

PURPOSE: The purpose of this study is to investigate a sol-gel transition property and content release profiles for thermosensitive poly-(D,L-lactide-co-glycolide)-block-poly-(ethylene glycol)-block-poly-(D,L-lactide-co-glycolide) (PLGA-b-PEG-b-PLGA) hydrogels carrying paclitaxel, rapamycin, and LS301, and to present a proof-of-concept that PLGA-b-PEG-b-PLGA hydrogels carrying paclitaxel, rapamycin, and LS301, called TheranoGel, exhibit excellent theranostic activity in peritoneal ES-2-luc ovarian cancer xenograft mice. METHODS: Thermosensitive PLGA-b-PEG-b-PLGA hydrogels carrying paclitaxel, rapamycin, and LS301, individually or in combination, were prepared via a lyophilization method, characterized with content release kinetics, and assessed with theranostic activity in ES-2-luc xenograft mice. RESULTS: A thermosensitive PLGA-b-PEG-b-PLGA sol-gel system was able to entrain 3 poorly water-soluble payloads, paclitaxel, rapamycin, and LS301 (TheranoGel). TheranoGel made a sol-to-gel transition at 37°C and slowly released 3 drugs at a simultaneous release rate in response to the physical dissociation of hydrogels in vitro. TheranoGel enabled loco-regional delivery of multi-drugs by forming a gel-depot in the peritoneal cavity of ES-2-luc xenograft mice. An intraperitoneal (IP) administration of TheranoGel resulted in excellent therapeutic and diagnostic activities, leading to the improved peritoneal surgery in ES-2-luc xenograft mice. CONCLUSIONS: TheranoGel prepared via a facile lyophiliation method enabled successful IP delivery of multi-drugs and exhibited excellent theranostic activity in vivo.

Polymeric micelles for multi-drug delivery in cancer.

Drug combinations are common in cancer treatment and are rapidly evolving, moving beyond chemotherapy combinations to combinations of signal transduction inhibitors. For the delivery of drug combinations, i.e., multi-drug delivery, major considerations are synergy, dose regimen (concurrent versus sequential), pharmacokinetics, toxicity, and safety. In this contribution, we review recent research on polymeric micelles for multi-drug delivery in cancer. In concurrent drug delivery, polymeric micelles deliver multi-poorly water-soluble anticancer agents, satisfying strict requirements in solubility, stability, and safety. In sequential drug delivery, polymeric micelles participate in pretreatment strategies that "prime" solid tumors and enhance the penetration of secondarily administered anticancer agent or nanocarrier. The improved delivery of multiple poorly water-soluble anticancer agents by polymeric micelles via concurrent or sequential regimens offers novel and interesting strategies for drug combinations in cancer treatment.

[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

Entropy-Driven Liquid-Liquid Phase Separation Transition to Polymeric Micelles.

In recent years, liquid-liquid phase separation (LLPS) has been recognized to act as a precursor to self-assembly in amphiphilic systems. In this study, we propose the use of entropy-driven LLPS to obtain a tunable precursor for polymeric micelle formation. In this new approach, an oligomer is utilized as a nonselective solvent for the block copolymer, allowing for the tuning of entropy and subsequent LLPS. A comprehensive model was developed using mean-field lattice theory to predict the conditions under which LLPS and micellization occur. The degree of polymerization of the solvent was found to have a significant impact on the phase behavior of the system, outweighing enthalpic contributions such as the interaction between the blocks of the copolymer and the solvent. Our model predicts that using a solvent with a degree of polymerization equal to or greater than 5 for a copolymer such as PEG4kDa-b-PLA2.2kDa will result in LLPS prior to complete micellization, regardless of the values of interaction parameters. It also suggests that phase-separated liquid and polymeric micelles can co-exist in such a mixture. We confirmed our model predictions using dynamic light scattering and phase microscopy when PEG200 was used as the solvent. Micellization for PEG4kDa-b-PLA2.2kDa/PEG200/water mixture occurred at 10-12% w/w water content, consistent with the model predictions. Furthermore, the LLPS-to-micelle transition was shown to be reversible by changing the temperature or water content, indicating that the phase-separated liquid may be in thermodynamic equilibrium with polymeric micelles.

Fibronectin-targeted FUD and PEGylated FUD peptides for fibrotic diseases.

Tissue fibrosis is characterized by excessive deposition of extracellular matrix (ECM) molecules. Fibronectin (FN) is a glycoprotein found in the blood and tissues, a key player in the assembly of ECM through interaction with cellular and extracellular components. Functional Upstream Domain (FUD), a peptide derived from an adhesin protein of bacteria, has a high binding affinity for the N-terminal 70-kDa domain of FN that plays a crucial role in FN polymerization. In this regard, FUD peptide has been characterized as a potent inhibitor of FN matrix assembly, reducing excessive ECM accumulation. Furthermore, PEGylated FUD was developed to prevent rapid elimination of FUD and enhance its systemic exposure in vivo. Herein, we summarize the development of FUD peptide as a potential anti-fibrotic agent and its application in experimental fibrotic diseases. In addition, we discuss how modification of the FUD peptide via PEGylation impacts pharmacokinetic profiles of the FUD peptide and can potentially contribute to anti-fibrosis therapy.

Plasma Stability and Plasma Metabolite Concentration-Time Profiles of Oligo(Lactic Acid)8-Paclitaxel Prodrug Loaded Polymeric Micelles.

Paclitaxel (PTX) is a frequently prescribed chemotherapy drug used to treat a wide variety of solid tumors. Oligo(lactic acid)8-PTX prodrug (o(LA)8-PTX) loaded poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) micelles have higher loading, slower release and higher antitumor efficacy in murine tumor models over PTX-loaded PEG-b-PLA micelles. The goal of this work is to study plasma stability of o(LA)8-PTX-loaded PEG-b-PLA micelles and its pharmacokinetics after IV injection in rats. In rat plasma, o(LA)8-PTX prodrug is metabolized into o(LA)1-PTX and PTX. In human plasma, o(LA)8-PTX is metabolized more slowly into o(LA)2-PTX, o(LA)1-PTX, and PTX. After IV injection of 10 mg/kg PTX-equiv of o(LA)8-PTX prodrug loaded PEG-b-PLA micelles in Sprague-Dawley rats, metabolite abundance in plasma follows the order: o(LA)1-PTX > o(LA)2-PTX > o(LA)4-PTX > o(LA)6-PTX. Bile metabolite profiles of the o(LA)8-PTX prodrug is similar to plasma metabolite profiles. In comparison to equivalent doses of Abraxane®, plasma PTX exposure is two orders of magnitude higher for Abraxane® than PTX from o(LA)8-PTX prodrug loaded PEG-b-PLA micelles, and plasma o(LA)1-PTX exposure is fivefold higher than PTX from Abraxane®, demonstrating heightened plasma metabolite exposure for enhanced antitumor efficacy.

In vivo cancer imaging by poly(ethylene glycol)-b-poly(ɛ-caprolactone) micelles containing a near-infrared probe.

Noninvasive near-infrared (NIR) fluorescence imaging is a promising technique for the intraoperative assessment of solid tumor removal. We incorporated a lipophilic NIR probe, 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR), in poly(ethylene glycol)-b-poly(ɛ-caprolactone) (PEG-b-PCL) micelles, resulting in DiR solubilization in water, occupying nanoscopic PEG-b-PCL micelles. DiR in a self-quenched or nonquenched state showed different kinetics of release from PEG-b-PCL micelles in vitro; however, both obtained high tumor delineation (tumor-to-muscle ratio of 30-43 from collected organs). These results suggest that PEG-b-PCL micelles with DiR are a promising nanosized imaging agent that will provide a basis for enhanced surgical guidance via NIR visualization of tumors. FROM THE CLINICAL EDITOR: In this paper, noninvasive near-infrared fluorescence imaging coupled with specific lipophilic probes is discussed as a promising technique for intraoperative assessment of solid tumor removal, leading to optimized outcomes for in toto removal of tumors.

Production of paclitaxel-loaded PEG-b-PLA micelles using PEG for drug loading and freeze-drying.

A new approach named PEG-assist is introduced for the production of drug-loaded polymeric micelles. The method is based on the use of PEG as the non-selective solvent for PEG-b-PLA in the fabrication procedure. Both hydration temperature and PEG molecular weight are shown to have a significant effect on the encapsulation efficiency of PTX in PEG4kDa-b-PLA2kDa micelles. The optimal procedure for fabrication includes the use of PEG1kDa as the solvent at 60 °C, cooling the mixture to 40 °C, hydration at 40 °C, freezing at -80 °C and freeze-drying at -35 °C, 15 Pa. No significant difference (p > 0.05) in PTX encapsulation, average particle size and polydispersity index is observed between the samples before freeze-drying and after reconstitution of the freeze-dried cake. The prepared PTX formulations are stable at room temperature for at least 8 h. Scaling the batch size to 25× leads to no significant change (p > 0.05) in PTX encapsulation, average particle size and polydispersity index. PEG-assist method is applicable to other drugs such as 17-AAG, and copolymers of varied molecular weights. The use of no organic solvent, simplicity, cost-effectiveness, and efficiency makes PEG-assist a very promising approach for large scale production of drug-loaded polymeric micelles.

Multimodal imaging demonstrates enhanced tumor exposure of PEGylated FUD peptide in breast cancer.

In breast cancer, the extracellular matrix (ECM) undergoes remodeling and changes the tumor microenvironment to support tumor progression and metastasis. Fibronectin (FN) assembly is an important step in the regulation of the tumor microenvironment since the FN matrix precedes the deposition of various other ECM proteins, controls immune cell infiltration, and serves as a reservoir for cytokines and growth factors. Therefore, FN is an attractive target for breast cancer therapy and imaging. Functional Upstream Domain (FUD) is a 6-kDa peptide targeting the N-terminal 70-kDa domain of FN, which is critical for fibrillogenesis. FUD has previously been shown to function as an anti-fibrotic peptide both in vitro and in vivo. In this work, we conjugated the FUD peptide with 20-kDa of PEG (PEG-FUD) and demonstrated its improved tumor exposure compared to non-PEGylated FUD in a murine breast cancer model via multiple imaging modalities. Importantly, PEG-FUD peptide retained a nanomolar binding affinity for FN and maintained in vitro plasma stability for up to 48 h. Cy5-labeled PEG-FUD bound to exogenous or endogenous FN assembled by fibroblasts. The in vivo fluorescence imaging with Cy5-labeled FUD and FUD conjugates demonstrated that PEGylation of the FUD peptide enhanced blood exposure after subcutaneous (SC) injection and significantly increased accumulation of FUD peptide in 4T1 mammary tumors. Intravital microscopy confirmed that Cy5-labeled PEG-FUD deposited mostly in the extravascular region of the tumor microenvironment after SC administration. Lastly, positron emission tomography/computed tomography imaging showed that 64Cu-labeled PEG-FUD preferentially accumulated in the 4T1 tumors with improved tumor uptake compared to 64Cu-labeled FUD (48 h: 1.35 ± 0.05 vs. 0.59 ± 0.03 %IA/g, P < 0.001) when injected intravenously (IV). The results indicate that PEG-FUD targets 4T1 breast cancer with enhanced tumor retention compared to non-PEGylated FUD, and biodistribution profiles of PEG-FUD after SC and IV injection may guide the optimization of PEG-FUD as a therapeutic and/or imaging agent for use in vivo.

A 3-in-1 polymeric micelle nanocontainer for poorly water-soluble drugs.

Poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) micelles have a proven capacity for drug solubilization and have entered phase III clinical trials as a substitute for Cremophor EL in the delivery of paclitaxel in cancer therapy. PEG-b-PLA is less toxic than Cremophor EL, enabling a doubling of paclitaxel dose in clinical trials. We show that PEG-b-PLA micelles act as a 3-in-1 nanocontainer for paclitaxel, 17-allylamino-17-demethoxygeldanamycin (17-AAG), and rapamycin for multiple drug solubilization. 3-in-1 PEG-b-PLA micelles were ca. 40 nm in diameter; dissolved paclitaxel, 17-AAG, and rapamycin in water at 9.0 mg/mL; and were stable for 24 h at 25 °C. The half-life for in vitro drug release (t(1/2)) for 3-in-1 PEG-b-PLA micelles was 1-15 h under sink conditions and increased in the order of 17-AAG, paclitaxel, and rapamycin. The t(1/2) values correlated with log P(o/w) values, implicating a diffusion-controlled mechanism for drug release. The IC(50) value of 3-in-1 PEG-b-PLA micelles for MCF-7 and 4T1 breast cancer cell lines was 114 ± 10 and 25 ± 1 nM, respectively; combination index (CI) analysis showed that 3-in-1 PEG-b-PLA micelles exert strong synergy in MCF-7 and 4T1 breast cancer cell lines. Notably, concurrent intravenous (iv) injection of paclitaxel, 17-AAG, and rapamycin using 3-in-1 PEG-b-PLA micelles was well-tolerated by FVB albino mice. Collectively, these results suggest that PEG-b-PLA micelles carrying paclitaxel, 17-AAG, and rapamycin will provide a simple yet safe and efficacious 3-in-1 nanomedicine for cancer therapy.

Acyl and oligo(lactic acid) prodrugs for PEG-b-PLA and PEG-b-PCL nano-assemblies for injection.

Poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) and poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) form nano-assemblies, including micelles and nanoparticles, that increase the water solubility of anticancer drugs for injection. PEG-b-PLA and PEG-b-PCL are less toxic than commonly used organic solvents or solubilizers for injection, such as Cremophor EL® in Taxol®. Formulating paclitaxel in PEG-b-PLA micelles, as Genexol-PM®, permits dose escalation over Taxol®, enhancing antitumor efficacy in breast, lung and ovarian cancers. To expand the repertoire of anticancer drugs for injection, acyl and oligo(lactic acid) ester prodrugs have been synthesized for PEG-b-PLA and PEG-b-PCL nano-assemblies, compatibility, and novel nanomedicines for injection. Notably, acyl and oligo(lactic acid) taxane prodrugs delivered by PEG-b-PLA and PEG-b-PCL nano-assemblies display heightened plasma exposure, reduction in biodistribution into major organs and enhanced tumor exposure in murine tumor models, versus parent anticancer drugs in conventional formulations. As a result, acyl and oligo(lactic acid) ester prodrugs are less toxic and induce durable antitumor responses. In summary, acyl and oligo(lactic acid) ester prodrugs widen the range of anticancer drugs that can be tested safely and effectively by using PEG-b-PLA and PEG-b-PCL nano-assemblies, and they display superior anticancer efficacy over parent anticancer drugs, which are often approved products. Oligo(lactic acid) ester taxane prodrugs are in pre-clinical development as novel drug combinations and immunotherapy combinations for cancer therapy.

Oligo(Lactic Acid)8-Docetaxel Prodrug-Loaded PEG-b-PLA Micelles for Prostate Cancer.

Docetaxel (DTX) is among the most frequently prescribed chemotherapy drugs and has recently been shown to extend survival in advanced prostate cancer patients. However, the poor water solubility of DTX prevents full exploitation of this potent anticancer drug. The current marketed formulation, Taxotere®, contains a toxic co-solvent that induces adverse reactions following intravenous injection. Nano-sized polymeric micelles have been proposed to create safer, water-soluble carriers for DTX, but many have failed to reach the clinic due to poor carrier stability in vivo. In this study, we aimed to improve micelle stability by synthesizing an ester prodrug of DTX, oligo(lactic acid)8-docetaxel (o(LA)8-DTX), for augmented compatibility with the core of poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) micelles. Due to the enhancement of drug-carrier compatibility, we were able to load 50% (w/w) prodrug within the micelle, solubilize 20 mg/mL o(LA)8-DTX (~12 mg/mL DTX-equivalent) in aqueous media, and delay payload release. While the micelle core prohibited premature degradation, o(LA)8-DTX was rapidly converted to parent drug DTX through intramolecular backbiting (t1/2 = 6.3 h) or esterase-mediated degradation (t1/2 = 2.5 h) following release. Most importantly, o(LA)8-DTX micelles proved to be as efficacious but less toxic than Taxotere® in a preclinical mouse model of prostate cancer.

The virucidal EB peptide protects host cells from herpes simplex virus type 1 infection in the presence of serum albumin and aggregates proteins in a detergent-like manner.

The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634-2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 µM concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 µM, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (≥0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for ß-galactosidase, EB below 20 µM acted as an enzyme inhibitor, whereas above 40 to 100 µM EB, ß-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins.

Enhanced stability of PEG-block-poly(N-hexyl stearate l-aspartamide) micelles in the presence of serum proteins.

Polyethylene glycol-phospholipid micelles form a major class of nanocarriers in pharmacy and medicine due to proven capability in drug solubilization, sustained drug release, and evidence for targeted drug delivery in vivo. In this report, we have prepared micelles composed of PEG-block-poly(N-hexyl stearate l-aspartamide) (PEG-b-PHSA), having nine stearic acid side chains, and have studied their stability in the presence of serum proteins by Forster resonance energy transfer (FRET) experiments. In the presence of serum albumin, alpha and beta globulins, or gamma globulins, there are minimal changes in FRET over two hours in vitro, indicating integrity of PEG-b-PHSA micelles. In contrast, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (PEG-DSPE) micelles lose FRET over two hours in vitro, especially in the presence of alpha and beta globulins, indicating the disruption of PEG-DSPE micelles and leakage of fluorescent probes. Owing to the aliphatic nature of DSPE and PHSA, both PEG-b-PHSA and PEG-DSPE micelles efficiently solubilize amphotericin B (AmB), a poorly water-soluble antifungal agent used to combat systemic mycoses. However, only PEG-b-PHSA micelles gradually liberate AmB in the presence of alpha and beta globulins, based on time-dependent changes in the self-aggregation state of AmB, monitored by UV/vis spectroscopy. PEG-b-PHSA micelles are remarkably stable in the presence of serum proteins and a more stable alternative for poorly water-soluble drugs, which have been solubilized by PEG-DSPE micelles.

pH-sensitive multi-PEGylated block copolymer as a bioresponsive pDNA delivery vector.

PURPOSE: A reversibly-PEGylated diblock copolymer, poly(aspartate-hydrazide-poly(ethylene glycol))-block-poly(aspartate-diaminoethane) (p[Asp(Hyd-PEG)]-b-p[Asp(DET)]) was reported here for enhanced gene transfection and colloidal stability. The diblock copolymer possessed a unique architecture based on a poly(aspartamide) backbone. The first block, p[Asp(Hyd)], was used for multi-PEG conjugations, and the second block, p[Asp(DET)], was used for DNA condensation and endosomal escape. METHODS: p[Asp(Hyd-PEG)]-b-p[Asp(DET)] was synthesized and characterized by (1)H-NMR. Polyplexes were formed by mixing the synthesized polymers and pDNA. The polyplex size, ζ-potential, and in vitro transfection efficiency were determined by dynamic light scattering, ζ-potential measurements, and luciferase assays, respectively. pH-dependent release of PEG from the polymer was monitored by cationic-exchange chromatography. RESULTS: The polyplexes were 70-90 nm in size, and the surface charge was effectively shielded by a PEG layer. The transfection efficiency of the reversibly PEGylated polyplexes was confirmed to be comparable to that of the non-PEGylated counterparts and 1,000 times higher than that of the irreversibly PEGylated polyplexes. PEG release was demonstrated to be pH-sensitive. Fifty percent of the PEG was released within 30 min at pH 5, while the polymer incubated at pH 7.4 could still maintain 50% of PEG after 8 h. CONCLUSION: The reversibly PEGylated polyplexes were shown to maintain polyplex stability without compromising transfection efficiency.