Polyethyleneimine grafted with pluronic P85 enhances Ku86 antisense delivery and the ionizing radiation treatment efficacy in vivo.

In an effort to improve the efficacy of antisense delivery, we evaluated polyethyleneimine (PEI, 2 kDa) alone or grafted with nonionic amphiphilic block copolymer Pluronic (P85) as a carrier for Ku86 antisense oligonucleotide (ASO) delivery. Ku86 is an abundant nuclear protein that plays an important role in nonhomologous DNA end joining and has implications in tumorigenesis and acquired drug resistance. Transfection of adherent and suspension cell lines with Ku86 ASOs complexed with P85-g-PEI (2 kDa) conjugates was associated with a specific decrease in Ku86 mRNA levels (EC50<75 nM and EC50<250 nM, respectively, n=3). More importantly, no requirement for reduced serum conditions was necessary during transfection. In contrast, whereas Ku86 ASOs complexed with PEI (2 kDa) alone were effective in decreasing Ku86 mRNA levels in adherent cell lines (EC50<75 nM, n=3), the formulation did not produce any detectable decrease in Ku86 mRNA levels in suspension cell lines. Transfection of adherent cell lines with 500 nM Ku86 ASOs formulated with P85-g-PEI (2 kDa) was associated with a specific decrease (<10% remaining of control) in Ku86 protein expression and a two-fold increased cell death after treatment with ionizing radiation (IR). In athymic nude mice bearing subcutaneous human HT29 colon adenocarcinoma xenografts, Ku86 ASO-P85-g-PEI (2 kDa) administration (15 mg/kg, subcutaneously) with a Q1D x 7 treatment schedule, when combined with a single dose of IR (6 Gy), caused a significant inhibition of HT29 tumor growth compared with mismatch- and naked antisense-pretreated control groups (time from 200 to 1000 mm3, 126.9 versus 84.18 and 87.76 days, P<0.005). A potentiation of the antitumor activity was observed in all mice treated with Ku86 ASO-P85-g-PEI (2 kDa) formulation; however, tumor growth inhibition was reversible upon treatment cessation. No morbidity/mortality or changes in histopathology were observed under this treatment regiment. Our results indicate that P85-g-PEI (2 kDa) conjugates may increase the efficacy of Ku86 ASO delivery in management of resistant malignancies, thus providing a rationale for their evaluation in cancer patients in combination with conventional anticancer therapies.

Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: Selective energy depletion.

This paper, for the first time, demonstrates that exposure of cells to the poly(ethylene oxide)-poly(propylene oxide) block copolymer, Pluronic P85, results in a substantial decrease in ATP levels selectively in MDR cells. Cells expressing high levels of functional P-glycoprotein (MCF-7/ADR, KBv; LLC-MDR1; Caco-2, bovine brain microvessel endothelial cells [BBMECs]) are highly responsive to Pluronic treatment, while cells with low levels of P-glycoprotein expression (MCF-7, KB, LLC-PK1, human umbilical vein endothelial cells [HUVECs] C2C12 myoblasts) are much less responsive to such treatment. Cytotoxicity studies suggest that Pluronic acts as a chemosensitizer and potentiates cytotoxic effects of doxorubicin in MDR cells. The ability of Pluronic to inhibit P-glycoprotein and sensitize MDR cells appears to be a result of ATP depletion. Because many mechanisms of drug resistance are energy dependent, a successful strategy for treating MDR cancer could be based on selective energy depletion in MDR cells. Therefore, the finding of the energy-depleting effects of Pluronic P85, in combination with its sensitization effects is of considerable theoretical and practical significance.

Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.

Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.

Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies.

Drug delivery across the blood-brain barrier is limited by several mechanisms. One important mechanism is drug efflux, mediated by several transport proteins, including P-glycoprotein. The goal of this work was to examine the effect of a novel drug delivery system, Pluronic block copolymer P85, on P-glycoprotein-mediated efflux from the brain using in vitro and in vivo methods. The hypothesis was that specific Pluronic copolymer systems enhance drug delivery to the central nervous system through the inhibition of P-glycoprotein. The effect of P85 on the cellular accumulation and transport of digoxin, a model P-glycoprotein substrate, was examined in porcine kidney epithelial cells (LLC-PK1) transfected with the human MDR1 gene. The effect of P85 on the directional flux across an in vitro BBB was also characterized. In vivo brain distribution studies were accomplished using wild-type and P-glycoprotein knockout mice. Pluronic increased the cellular accumulation of digoxin 3-fold in LLC-PK1 cells and 5-fold in the LLC-PK1-MDR1-transfected cells. Similar effects were observed for a prototypical P-glycoprotein substrate rhodamine-123. P85 treatment decreased the basolateral-to-apical and increased the apical-to-basolateral digoxin flux across LLC-PK1-MDR1 cell monolayers, and analogous results were observed with the in vitro BBB monolayers. The coadministration of 1% P85 with radiolabeled digoxin in wild-type mice increased the brain penetration of digoxin 3-fold and the digoxin level in the P85-treated wild-type mice was similar to that observed in the P-glycoprotein-deficient animals. These data indicate that Pluronic P85 can enhance the delivery of digoxin to the brain through the inhibition of the P-glycoprotein-mediated efflux mechanism.

Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell.

Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic. block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes. which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes ("stealth effect"). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mixing with NanoGel suspension, which results in the formation of small particles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion. Further. loaded NanoGel particles cross-polarized monolayers of intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents.

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO-PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.

Block copolymeric biotransport carriers as versatile vehicles for drug delivery.

This review describes block co-polymer-based systems that are used in drug delivery. The main focus is on amphiphilic block co-polymers, the application of which modifies the pharmacological performance of various classes of drugs and is attracting more and more attention. The two main reasons for this are the high tendency of block co-polymer-based drug formulations to self-assemble and the flexibility of block co-polymer chemistry, which allows precise tailoring of the carrier to virtually any chemical entity. The combination of these and some other features makes it possible to adjust block co-polymer-based drug formulations to achieve the most beneficial balance in their biological interactions (biotransport), with systems that control drug removal from the body and those that are responsible for drug therapeutic activity. The following major aspects are considered: The role of physical properties of formulations in their pharmacological performance. The chemistry and physico-chemistry of block co-polymers and structure-function relationships in these systems. Examples describing the effects of biotransport systems on drug transport and activity in cells and some results on their in vivo applications with various drugs.

Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity.

The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently (Alakhov VY, Moskaleva EY, Batrakova EV, Kabanov AV 1996, Biocon. Chem., 7, 209). This paper presents initial studies on in vivo evaluation of Pluronic copolymers in the treatment of cancer. The anti-tumour activity of epirubicin (EPI) and doxorubicin (DOX), solubilised in micelles of Pluronic L61, P85 and F108, was investigated using murine leukaemia P388 and daunorubicin-sensitive Sp2/0 and -resistant Sp2/0(DNR) myeloma cells grown subcutaneously (s.c.). The study revealed that the lifespan of the animals and inhibition of tumour growth were considerably increased in mice treated with drug/copolymer compositions compared with animals treated with the free drugs. The anti-tumour activity of the drug/copolymer compositions depends on the concentration of the copolymer and its hydrophobicity, as determined by the ratio of the lengths of hydrophilic PEO and hydrophobic PPO segments. The data suggest that higher activity is associated with more hydrophobic copolymers. In particular, a significant increase in lifespan (T/C> 150%) and tumour growth inhibition (> 90%) was observed in animals with Sp2/0 tumours with EPI/P85 and DOX/L61 compositions. The effective doses of these compositions caused inhibition of Sp2/0 tumour growth and complete disappearance of tumour in 33-50% of animals. Future studies will focus on the evaluation of the activity of Pluronic-based compositions against human drug-resistant tumours.