Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Pharmacokinetics of small hyperbranched polyglycerols as an osmotic agent for peritoneal dialysis: Plasma exposure, organ distribution and excretion in rats.

BACKGROUND: Small hyperbranched polyglycerol (HPG) has been recently of interest for peritoneal dialysis, but its pharmacokinetics is barely understood. This study investigated the absorption, distribution and excretion of 1 and 3 kDa HPG. METHODS: Rats (naive, 5/6 nephrectomy (5/6 Nx) or bilateral nephrectomy (BNx)) received a single dose of 3H-labelled HPG-containing solutions intraperitoneally (IP) or intravenously (IV). Radioactivity in tissues, urine and faeces was counted using a scintillation counter. Pharmacokinetic parameters were calculated using WinNonlin software. RESULTS: During 8-h dwell with IP injected therapeutic dose of HPG-based hypertonic solutions, the plasma levels of 1 kDa HPG reached the peak at 2 h, followed by a decrease to the end, whereas 3 kDa HPG increased for the duration of the 8 h. At the experimental endpoint, the distribution of both sizes of HPG in major organs was minimal, whereas most of 1 kDa HPG was excreted via urine, and of 3 kDa remained in peritoneal cavity. The elimination of both 1 and 3 kDa HPG after either IP or IV administration was significantly delayed by 5/6 Nx or BNx as compared to naive controls. Further, 24-h faecal excretion of HPG (3 kDa) was <5% of injected dose that was not different between healthy and BNx rats. CONCLUSION: Data suggest size-dependent peritoneal absorption of osmotic HPG that are not specifically absorbed by any of the organs tested. The clearance of small HPG mainly depends on kidney excretion, implying the risk of HPG accumulation in patients with end-stage kidney disease who receive maintenance dialysis with HPG.

Transcriptome analysis of signaling pathways of human peritoneal mesothelial cells in response to different osmotic agents in a peritoneal dialysis solution.

BACKGROUND: Glucose is a primary osmotic agent in peritoneal dialysis (PD) solutions, but its long-term use causes structural alteration of the peritoneal membrane (PM). Hyperbranched polyglycerol (HPG) is a promising alternative to glucose. This study was designed to compare the cellular responses of human peritoneal mesothelial cells (HPMCs) to these two different osmotic agents in a hypertonic solution using transcriptome analysis. METHODS: Cultured HPMCs were repeatedly exposed to HPG-based or Physioneal 40 (PYS, glucose 2.27%) hypertonic solutions. Transcriptome datasets were produced using Agilent SurePrint G3 Human GE 8 × 60 microarray. Cellular signaling pathways were examined by Ingenuity Pathway Analysis (IPA). Protein expression was examined by flow cytometry analysis and Western blotting. RESULTS: The HPG-containing solution was better tolerated compared with PYS, with less cell death and disruption of cell transcriptome. The levels of cell death in HPG- or PYS- exposed cells were positively correlated with the number of affected transcripts (HPG: 128 at day 3, 0 at day 7; PYS: 1799 at day 3, 212 at day 7). In addition to more affected "biosynthesis" and "cellular stress and death" pathways by PYS, both HPG and PYS commonly affected "sulfate biosynthesis", "unfolded protein response", "apoptosis signaling" and "NRF2-mediated oxidative stress response" pathways at day 3. PYS significantly up-regulated HLA-DMB and MMP12 in a time-dependent manner, and stimulated T cell adhesion to HPMCs. CONCLUSION: The lower cytotoxicity of hypertonic HPG solution is in agreement with its transient and minimal impact on the pathways for the "biosynthesis of cell constituents" and the "cellular stress and death". The significant up-regulation of HLA-DMB and MMP12 by PYS may be part of its initiation of immune response in the PM.

Peritoneal and Systemic Responses of Obese Type II Diabetic Rats to Chronic Exposure to a Hyperbranched Polyglycerol-Based Dialysis Solution.

Metabolic syndrome (MetS) is commonly observed among peritoneal dialysis (PD) patients, and hyperbranched polyglycerol (HPG) is a promising glucose-sparing osmotic agent for PD. However, the biocompatibility of a HPG-based PD solution (HPG) in subjects with MetS has not been investigated. This study compared the local and systemic effects of a HPG solution with conventional physioneal (PYS) and icodextrin (ICO) PD solutions in rats with MetS. Obese type 2 diabetic ZSF1 rats received a daily intraperitoneal injection of PD solutions (10 mL) for 3 months. The peritoneal membrane (PM) function was determined by ultrafiltration (UF), and the systemic responses were determined by profiling blood metabolic substances, cytokines and oxidative status. Tissue damage was assessed by histology. At the end of the 3-month treatment with PD solutions, PM damage and UF loss in both the PYS and ICO groups were greater than those in the HPG group. Blood analyses showed that compared to the baseline control, the rats in the HPG group exhibited a significant decrease only in serum albumin and IL-6 and a minor glomerular injury, whereas in both the PYS and ICO groups, there were more significant decreases in serum albumin, antioxidant activity, IL-6, KC/GRO (CXCL1) and TNF-α (in ICO only) as well as a more substantial glomerular injury compared to the HPG group. Furthermore, PYS increased serum creatinine, serum glucose and urine production. In conclusion, compared to PYS or ICO solutions, the HPG solution had less adverse effects locally on the PM and systemically on distant organs (e.g. kidneys) and the plasma oxidative status in rats with MetS.

Nontransformed and Cancer Cells Can Utilize Different Endocytic Pathways To Internalize Dendritic Nanoparticle Variants: Implications on Nanocarrier Design.

Three hyperbranched polyglycerol nanoparticle (HPG NP) variants were synthesized and fluorescently labeled for the study of their cellular interactions. The polymeric nanoparticle that contains a hydrophobic core and a hydrophilic HPG shell, HPG-C10-HPG, is taken up faster by HT-29 cancer cells than nontransformed cells, while similar uptake rates are observed with both cell types for the nanoparticle HPG-C10-PEG that contains a hydrophobic core and a polyethylene glycol shell. The nanoparticle HPG-104, containing neither the hydrophobic core nor the polyethylene glycol shell, is taken up faster by nontransformed cells than HT-29 cells. Importantly, cancer and normal cells can utilize different endocytic mechanisms for the internalization of these HPG NPs. Both HPG-C10-HPG and HPG-C10-PEG are taken up by HT-29 cells through clathrin-mediated endocytosis and macropinocytosis. Nontransformed cells, however, take up HPG-C10-HPG and HPG-104 through macropinocytosis, while these cells utilize both clathrin-mediated endocytosis and macropinocytosis to internalize HPG-C10-PEG.

Cold preservation with hyperbranched polyglycerol-based solution improves kidney functional recovery with less injury at reperfusion in rats.

Minimizing donor organ injury during cold preservation (including cold perfusion and storage) is the first step to prevent transplant failure. We recently reported the advantages of hyperbranched polyglycerol (HPG) as a novel substitute for hydroxyethyl starch in UW solution for both cold heart preservation and cold kidney perfusion. This study evaluated the functional recovery of the kidney at reperfusion after cold preservation with HPG solution. The impact of HPG solution compared to conventional UW and HTK solutions on tissue weight and cell survival at 4°C was examined using rat kidney tissues and cultured human umbilical vein endothelial cells (HUVECs), respectively. The kidney protection by HPG solution was tested in a rat model of cold kidney ischemia-reperfusion injury, and was evaluated by histology and kidney function. Here, we showed that preservation with HPG solution prevented cell death in cultured HUVECs and edema formation in kidney tissues at 4°C similar to UW solution, whereas HTK solution was less effective. In rat model of cold ischemia-reperfusion injury, the kidneys perfused and subsequently stored 1-hour with cold HPG solution showed less leukocyte infiltration, less tubular damage and better kidney function (lower levels of serum creatinine and blood urea nitrogen) at 48 h of reperfusion than those treated with UW or HTK solution. In conclusion, our data show the superiority of HPG solution to UW or HTK solution in the cold perfusion and storage of rat kidneys, suggesting that the HPG solution may be a promising candidate for improved donor kidney preservation prior to transplantation.

Hyperbranched polyglycerol is superior to glucose for long-term preservation of peritoneal membrane in a rat model of chronic peritoneal dialysis.

BACKGROUND: Replacing glucose with a better biocompatible osmotic agent in peritoneal dialysis (PD) solutions is needed in PD clinic. We previously demonstrated the potential of hyperbranched polyglycerol (HPG) as a replacement for glucose. This study further investigated the long-term effects of chronic exposure to HPG as compared to a glucose-based conventional PD solution on peritoneal membrane (PM) structure and function in rats. METHODS: Adult male Wistar rats received once-daily intraperitoneal injection of 10 mL of HPG solution (1 kDa, HPG 6%) compared to Physioneal™ 40 (PYS, glucose 2.27%) or electrolyte solution (Control) for 3 months. The overall health conditions were determined by blood chemistry analysis. The PM function was determined by ultrafiltration, and its injury by histological and transcriptome-based pathway analyses. RESULTS: Here, we showed that there was no difference in the blood chemistry between rats receiving the HPG and the Control, while PYS increased serum alkaline phosphatase, globulin and creatinine and decreased serum albumin. Unlike PYS, HPG did not significantly attenuate PM function, which was associated with smaller change in both the structure and the angiogenesis of the PM and less cells expressing vascular endothelial growth factor, α-smooth muscle actin and MAC387 (macrophage marker). The pathway analysis revealed that there were more inflammatory signaling pathways functioning in the PM of PYS group than those of HPG or Control, which included the signaling for cytokine production in both macrophages and T cells, interleukin (IL)-6, IL-10, Toll-like receptors, triggering receptor expressed on myeloid cells 1 and high mobility group box 1. CONCLUSIONS: The results from this experimental study indicate the superiority of HPG to glucose in the preservation of the peritoneum function and structure during the long-term PD treatment, suggesting the potential of HPG as a novel osmotic agent for PD.

Design Considerations for Developing Hyperbranched Polyglycerol Nanoparticles as Systemic Drug Carriers.

PEGylation is commonly used to increase the plasma residence time of anticancer drug nanocarriers. However, PEGylation may trigger antibody production and lead to accelerated blood clearance in subsequent administrations. Moreover, the presence of PEG shells on nanocarriers may also hamper endosomal escape and decrease drug payload release. To avoid these shortcomings, we synthesized and evaluated a non-PEGylated, hyperbranched polyglycerol nanoparticle (HPG NP) with a hydrophobic core and a hydrophilic HPG shell, HPG-C10-HPG, as a candidate for systemic delivery of anticancer drug. In vitro studies with primary human cell lines revealed that HPG-C10-HPG possesses low cytotoxicity. The presence of long chain alkyl groups (C1o) in the core as the hydrophobic pocket in the NP enabled the binding and sustained release of the hydrophobic drug docetaxel. Remarkably, the docetaxel-loaded HPG-C10-HPG formulation also confers preferential protection to primary cells, when compared to cancer cells, potentially widening the therapeutic index. HPG-C10-HPG, however, accumulated at higher levels in the liver and spleen when administered intravenously in mice. Comparing the biodistribution patterns of HPG-C10-HPG, PEGylated HPG-C10-PEG, and unmodified HPG in a xenograft model reveals that the accumulation pattern of HPG-C10-HPG was attributed to insufficient shielding of the hydrophobic groups by the HPG shell. Our results revealed the influence of the nature of the hydrophilic shell and the presence of hydrophobic groups on the tumor-to-tissue accumulation specificities of these HPG NP variants. Therefore, the present study provides insights into the structural considerations of future HPG NP designs for systemic drug delivery.

Investigation of hydrophobically derivatized hyperbranched polyglycerol with PEGylated shell as a nanocarrier for systemic delivery of chemotherapeutics.

We report the synthesis and characterization of a polymeric nanoparticle (NP) based on hyperbranched polyglycerol (HPG) containing a hydrophobic core and a hydrophilic shell, and assessed its suitability to be developed as a systemic anticancer drug carrier. HPG NP displayed low toxicity to primary cell cultures and were well-tolerated in mice after intravenous administration. When tested in mice tumor xenograft models, HPG NP accumulated significantly in the tumors with low accumulation in the liver and the spleen. In vitro studies demonstrated that HPG NP was capable of hydrophobically binding docetaxel and releasing it in a controlled manner. The HPG NP formulation of docetaxel conferred a preferential protective effect on primary non-cancerous cells while effectively killing cancer cells, indicating great potential for widening its therapeutic index. Taken together, these data indicate that HPG NP is a highly promising nanocarrier platform for systemic delivery of anticancer drugs. FROM THE CLINICAL EDITOR: The use of polyethylene glycol on nano-carriers as "stealth" to deliver intravenous drugs is well known. Here, the authors developed polymeric nanoparticle (NP) with hyperbranched polyglycerol (HPG) and tested its efficacy in delivering docetaxel. The results showed that this formulation could preferentially killed cancer cells with a high therapeutic index. It seems that this platform could have a great potential in cancer therapy.

Hyperbranched polyglycerol as a colloid in cold organ preservation solutions.

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

The size-dependent efficacy and biocompatibility of hyperbranched polyglycerol in peritoneal dialysis.

Glucose is a common osmotic agent for peritoneal dialysis (PD), but has many adverse side effects for patients with end-stage renal disease. Recently, hyperbranched polyglycerol (HPG) has been tested as an alternative osmotic agent for PD. This study was designed to further examine the efficacy and biocompatibility of HPG over a range of different molecular weights. HPGs of varying molecular weights (0.5 kDa, 1 kDa, 3 kDa) were evaluated in a preclinical rodent model of PD. HPG PD solutions were standardized for osmolality and compared directly to conventional glucose-based Physioneal™ PD solution (PYS). The efficacy of HPG solutions was measured by their ultrafiltration (UF) capacity, solute removal, and free water transport; biocompatibility was determined in vivo by the histological analysis of the peritoneal membrane and the cell count of detached peritoneal mesothelial cells (PMCs) and neutrophils, and in vitro cytotoxicity to cultured human PMCs. All the different sized HPGs induced higher UF and sodium removal over a sustained period of time (up to 8 h) compared to PYS. Urea removal was significantly higher for 1-3 kDa than PYS, and was similar for 0.5 kDa. Our analyses indicated that the peritoneal membrane exhibited more tolerance to the HPG solutions compared to PYS, evidenced by less submesothelial injury and neutrophil infiltration in vivo, and less cell death in cultured human peritoneal mesothelial cells. Free water transport analysis of HPG indicated that these molecules function as colloids and induce osmosis mainly through capillary small pores. We attribute the differences in the biocompatibility and osmotic activity of different sized HPGs to the differences in the polymer bound water measured by differential scanning calorimetry. These preclinical data indicate that compared to PYS, low MW HPGs (0.5-3 kDa) produces superior fluid and waste removal with better biocompatibility profile, suggesting that they are promising osmotic agents for PD.

Hyperbranched polyglycerol is an efficacious and biocompatible novel osmotic agent in a rodent model of peritoneal dialysis.

OBJECTIVES: To enhance the effectiveness of peritoneal dialysis (PD), new biocompatible PD solutions may be needed. The present study was designed to test the efficacy and biocompatibility of hyperbranched polyglycerol (HPG)-a nontoxic, nonimmunogenic water-soluble polyether polymer-in PD. METHODS: Adult Sprague-Dawley rats were instilled with 30 mL HPG solution (molecular weight 3 kDa; 2.5% - 15%) or control glucose PD solution (2.5% Dianeal: Baxter Healthcare Corporation, Deerfield, IL, USA), and intraperitoneal fluid was recovered after 4 hours. Peritoneal injury and cellular infiltration were determined by histologic and flow cytometric analysis. Human peritoneal mesothelial cells were assessed for viability in vitro after 3 hours of PD fluid exposure. RESULTS: The 15% HPG solution achieved a 4-hour dose-related ultrafiltration up to 43.33 ± 5.24 mL and a dose-related urea clearance up to 39.17 ± 5.21 mL, results that were superior to those with control PD solution (p < 0.05). The dialysate-to-plasma (D/P) ratios of urea with 7.5% and 15% HPG solution were not statistically different from those with control PD solution. Compared with fluid recovered from the control group, fluid recovered from the HPG group contained proportionally fewer neutrophils (3.63% ± 0.87% vs 9.31% ± 2.89%, p < 0.0001). Detachment of mesothelial cells positive for human bone marrow endothelial protein 1 did not increase in the HPG group compared with the stain control (p = 0.1832), but it was elevated in the control PD solution group (1.62% ± 0.68% vs 0.41% ± 0.31%, p = 0.0031). Peritoneal biopsies from animals in the HPG PD group, compared with those from control PD animals, demonstrated less neutrophilic infiltration and reduced thickness. Human peritoneal mesothelial cell survival after HPG exposure was superior in vitro (p < 0.0001, 7.5% HPG vs control; p < 0.01, 15% HPG vs control). Exposure to glucose PD solution induced cytoplasmic vacuolation and caspase 3-independent necrotic cell death that was not seen with HPG solution. CONCLUSIONS: Our novel HPG PD solution demonstrated effective ultrafiltration and waste removal with reduced peritoneal injury in a rodent model of PD.

Polyvalent choline phosphate as a universal biomembrane adhesive.

Phospholipids in the cell membranes of all eukaryotic cells contain phosphatidyl choline (PC) as the headgroup. Here we show that hyperbranched polyglycerols (HPGs) decorated with the 'PC-inverse' choline phosphate (CP) in a polyvalent fashion can electrostatically bind to a variety of cell membranes and to PC-containing liposomes, the binding strength depending on the number density of CP groups per macromolecule. We also show that HPG-CPs can cause cells to adhere with varying affinity to other cells, and that binding can be reversed by subsequent exposure to low molecular weight HPGs carrying small numbers of PCs. Moreover, PC-rich membranes adsorb and rapidly internalize fluorescent HPG-CP but not HPG-PC molecules, which suggests that HPG-CPs could be used as drug-delivery agents. CP-decorated polymers should find broad use, for instance as tissue sealants and in the self-assembly of lipid nanostructures.

In vitro and in vivo evaluation of intravesical docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols in an orthotopic model of bladder cancer.

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.