Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model.

Cerebral palsy (CP) is a chronic childhood disorder with no effective cure. Neuroinflammation, caused by activated microglia and astrocytes, plays a key role in the pathogenesis of CP and disorders such as Alzheimer's disease and multiple sclerosis. Targeting neuroinflammation can be a potent therapeutic strategy. However, delivering drugs across the blood-brain barrier to the target cells for treating diffuse brain injury is a major challenge. We show that systemically administered polyamidoamine dendrimers localize in activated microglia and astrocytes in the brain of newborn rabbits with CP, but not healthy controls. We further demonstrate that dendrimer-based N-acetyl-l-cysteine (NAC) therapy for brain injury suppresses neuroinflammation and leads to a marked improvement in motor function in the CP kits. The well-known and safe clinical profile for NAC, when combined with dendrimer-based targeting, provides opportunities for clinical translation in the treatment of neuroinflammatory disorders in humans. The effectiveness of the dendrimer-NAC treatment, administered in the postnatal period for a prenatal insult, suggests a window of opportunity for treatment of CP in humans after birth.

Injectable PAMAM dendrimer-PEG hydrogels for the treatment of genital infections: formulation and in vitro and in vivo evaluation.

Local intravaginal drug therapy is preferred for treatment of ascending genital infections during pregnancy. In the present study, an in situ forming biodegradable hydrogel for sustained release of amoxicillin in the cervicovaginal region is described. A generation 4 poly(amidoamine) [G4-(NH(2))(64)] dendrimer with peripheral thiopyridyl terminations is cross-linked with 8-arm polyethylene glycol (PEG) bearing thiol terminations. The hydrogels were formulated and tested in vivo in a pregnant guinea pig model for volume, retention times, biodegradation, tolerability and transport across fetal membrane. The physicochemical characterization of the hydrogels was carried out using differential calorimetry, SEM, and confocal imaging. The hydrogels offer antibacterial activity arising from sustained release of amoxicillin from gels. The in vivo studies in guinea pig showed that 100-200 µL of gel sufficiently covered the cervicovaginal region with a residence time of at least 72 h and gel was primarily retained in the maternal tissues without crossing the fetal membranes into the fetus. The dendrimer gels were stable up to 72 h, and the in vivo biodegradation of gel occurred after 72 h; this correlated well with the in vitro degradation pattern. The pH of the vagina was not altered upon application of the gel, and none of the animals aborted up to 72 h after application of gel. The histological evaluation of the cervical tissues showed absence of edema in the epithelial cell layer, no sloughing of the epithelial or superficial mucous layer, and absence of necrosis and infiltration of inflammatory cells in the submucosal layers, confirming that tissues were tolerant to the gel. The immunohistofluorescence images showed the localization of the gel components on the superficial mucified epithelial layer. The cross-linking density and swelling of hydrogels was impacted by the polymer content, and the 10% hydrogels exhibited the highest cross-link density. The in vitro drug release studies carried out using Franz diffusion cells showed that amoxicillin release from 6 and 10% gels was sustained for 240 h as compared to 3% gels. As the polymer concentration increased to 10%, the release pattern from gels approached diffusion controlled mechanism with diffusional exponent n = 0.49. In conclusion, the biodegradable in situ forming hydrogels of the present study offer a therapeutic option to provide sustained localized delivery of amoxicillin intracervically to the pregnant woman for the treatment of ascending genital infections.

Transfer of PAMAM dendrimers across human placenta: prospects of its use as drug carrier during pregnancy.

Dendrimers offer significant potential as nanocarriers for targeted delivery of drugs and imaging agents. The objectives of this study were to evaluate the transplacental transport, kinetics and biodistribution of PAMAM dendrimers ex-vivo across the human placenta in comparison with antipyrine, a freely diffusible molecule, using dually perfused re-circulating term human placental lobules. The purpose of this study is to determine if dendrimers as drug carriers can be used to design drug delivery systems directed at selectively treating either the mother or the fetus. The transplacental transfers of fluorescently (Alexa 488) tagged PAMAM dendrimer (16 kDa) and antipyrine (188 Da) from maternal to fetal circulation were measured using HPLC/dual UV and fluorescent detector (sensitivity of 10 ng/mL for dendrimer and 100 ng/mL for antipyrine respectively). C(max) for the dendrimer-Alexa (DA) in maternal perfusate (T(max)=15 min) was 18 times higher than in the fetal perfusate and never equilibrated with the maternal perfusate during 5.5 h of perfusion (n=4). DA exhibited a measurable but low transplacental transport of 2.26±0.12 µg/mL during 5.5h, where the mean transplacental transfer was 0.84±0.11% of the total maternal concentration and the feto-maternal ratio as percent was 0.073%±0.02. The biochemical and physiological analysis of the placentae perfused with DA demonstrated normal function throughout the perfusion. The immunofluorescence histochemistry confirmed that the biodistribution of DA in perfused placenta was sparsely dispersed, and when noted was principally seen in the inter-villous spaces and outer rim of the villous branches. In a few cases, DA was found internalized and localized in nuclei and cytoplasm of syncytiotrophoblast and inside the villous core; however, DA was mostly absent from the villous capillaries. In conclusion, the PAMAM dendrimers exhibited a low rate of transfer from maternal to the fetal side across the perfused human placenta, which is similar to other investigations of large macromolecules, e.g., IgG. These overall findings suggest that entry of drugs conjugated to polymers, i.e., dendrimers, would be limited in their transfer across the human placenta when compared to smaller drug molecules alone, suggesting novel methods for selectively delivering therapeutics to the pregnant woman without significant transfer to the fetus, especially since the half life of the dendrimer in blood is relatively short.

Intrinsic targeting of inflammatory cells in the brain by polyamidoamine dendrimers upon subarachnoid administration.

AIM: Understanding the interactions between nanomaterials and disease processes is crucial for designing effective therapeutic approaches. This article explores the unusual neuroinflammation targeting of dendrimers (with no targeting ligands) in the brain, with significant consequences for nanoscale materials in medicine. METHOD: The in vivo biodistribution of fluorescent-labeled neutral generation-4- polyamidoamine dendrimers (∼4 nm) in a rabbit model of cerebral palsy was explored following subarachnoid administration. RESULTS: These dendrimers, with no targeting ligands, were localizing in activated microglia and astrocytes (cells responsible for neuroinflammation), even in regions far moved from the site of injection, in newborn rabbits with maternal inflammation-induced cerebral palsy. CONCLUSION: This intrinsic ability of dendrimers to localize inactivated microglia and astrocytes can enable targeted delivery of therapeutics in disorders such as cerebral palsy, Alzheimer's and multiple sclerosis.

Inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using PAMAM dendrimers.

Dendrimers have emerged as topical microbicides to treat vaginal infections. This study explores the in vitro, in vivo antimicrobial activity of PAMAM dendrimers, and the associated mechanism. Interestingly, topical cervical application of 500 microg of generation-4 neutral dendrimer (G(4)-PAMAM-OH) showed potential to treat the Escherichia coli induced ascending uterine infection in guinea pig model of chorioamnionitis. Amniotic fluid collected from different gestational sacs of infected guinea pigs posttreatment showed absence of E. coli growth in the cultures plated with it. The cytokine level [tumor necrosis factor (TNFalpha) and interleukin (IL-6 and IL-1beta)] in placenta of the G(4)-PAMAM-OH treated animals were comparable to those in healthy animals while these were notably high in infected animals. Since, antibacterial activity of amine-terminated PAMAM dendrimers is known, the activity of hydroxyl and carboxylic acid terminated PAMAM dendrimers was compared with it. Though the G(4)-PAMAM-NH(2) shows superior antibacterial activity, it was found to be cytotoxic to human cervical epithelial cell line above 10 microg/mL, while the G(4)-PAMAM-OH was non-cytotoxic up to 1mg/mL concentration. Cell integrity, outer (OM) and inner (IM) membrane permeabilization assays showed that G(4)-PAMAM-OH dendrimer efficiently changed the OM permeability, while G(4)-PAMAM-NH(2) and G(3.5)-PAMAM-COOH damaged both OM and IM causing the bacterial lysis. The possible antibacterial mechanism are G(4)-PAMAM-NH(2) acts as polycation binding to the polyanionic lipopolysaccharide in E. coli, the G(4)-PAMAM-OH forms hydrogen bonds with the hydrophilic O-antigens in E. coli membrane and the G(3.5)-PAMAM-COOH acts as a polyanion, chelating the divalent ions in outer cell membrane of E. coli. This is the first study which shows that G(4)-PAMAM-OH dendrimer acts as an antibacterial agent.

Amino acid-functionalized dendrimers with heterobifunctional chemoselective peripheral groups for drug delivery applications.

Dendrimers have emerged as multifunctional carriers for targeted drug delivery, gene delivery and imaging. Improving the functional versatility at the surface for carrying multiple conjugation reactions is becoming vital. Typically, generation four polyamidoamine (G4-PAMAM) dendrimers bear approximately 64 symmetrical end groups, often requiring different spacers to conjugate various functional groups (drugs and targeting moities), increasing the synthetic steps. In the present study, a simple one-step synthesis to convert each symmetrical end group of G4-PAMAM dendrimers into two reactive, distinct orthogonal and chemoselective groups is described. A near-complete end-capping of the dendrimers (87-93%) with amino acids results in heterobifunctional G4-PAMAM dendrimers bearing a very high (> or = 110) diverse peripheral end groups (OH+NHBoc, OH+COOMe, SH+NHBoc, and COOH+NHBoc). Postfunctionalization ability of these dendrimers was evaluated. The heterobifunctional groups at the dendrimer periphery could be chemoselectively conjugated to multiple moities such as drugs (indomethacin and dexamethasone) and drugs and imaging agents (dexamethasone and FITC). These conjugations could be achieved in immediate succession without functional group conversions, eliminating the additional elaborate synthetic steps traditionally required to append specific linkers. Furthermore, one of the two functional handles at periphery was used to develop in situ forming hydrogels, whereas the other handle could be used for conjugating the drugs (e.g., dexamethasone). The heterobifunctional dendrimers with either "NH(2) or SH (thiopyridyl protected form)" terminations showed in situ hydrogel formation by cross-linking with N-hydroxysuccinimide or thiol-terminated multiarm polyethylene glycol (20 kDa). The choice of amino acids as versatile linkers would enable biocompatible dendrimer scaffolds for use in drug delivery. Zeta-potential measurements showed drastic lowering of the charge on G4-PAMAM-NH(2) dendrimers by end-capping with amino acids, whereas in the case of neutral G4-PAMAM-OH dendrimers, the charge did not increase or decrease substantially. The in vitro cytotoxicity and hemolysis assay showed that the heterobifunctional dendrimers were noncytotoxic in the 100 ng/mL to 1 mg/mL concentration range. With this study, we demonstrate the development of biocompatible dendrimers bearing multiple orthogonal surface groups, enabling the attachment of drugs, imaging agents, and gel formation using minimal synthetic steps.

Transport and biodistribution of dendrimers across human fetal membranes: implications for intravaginal administration of dendrimer-drug conjugates.

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size approximately 400 Da) and fluorophore-tagged G(4)-PAMAM dendrimers (approximately 16 kDa). The fluorophore-tagged G(4)-PAMAM dendrimers were synthesized and characterized using (1)H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than <3%, whereas the transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 x 10(-7) cm(2)/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 x 10(-8) cm(2)/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5-4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother without affecting the fetus.

Stimuli-responsive star poly(ethylene glycol) drug conjugates for improved intracellular delivery of the drug in neuroinflammation.

N-Acetyl cysteine (NAC) is a vital drug currently under clinical trials for the treatment of neuroinflammation in maternal-fetal applications. The free sulfhydryl groups in NAC lead to high plasma protein binding, resulting in low bioavailability. Preparation and activity of conjugates of NAC with thiol terminated multi-arm (6 and 8) poly(ethylene-glycol) (PEG) with disulfide linkages involving sulfhydryls of NAC are reported. Multiple copies (5 and 7) of NAC were conjugated on 6 and 8-arm-PEG respectively. Both the conjugates released 74% of NAC within 2h by thiol exchange reactions in the redox environment provided by glutathione (GSH) intracellularly (2-10mM). At physiological extracellular glutathione concentration (2 microM) both the conjugates were stable and did not release NAC. MTT assay showed comparable cell viability for unmodified PEGs and both the PEG-S-S-NAC conjugates. The conjugates were readily endocytosed by cells, as confirmed by flow cytometry and confocal microscopy. Efficacy of 6 and 8-arm-PEG-S-S-NAC conjugates was evaluated on activated microglial cells (the target cells, in vivo) by monitoring cytokine release in lipopolysaccharide (LPS) induced inflammatory response in microglial cells using the reactive oxygen species (ROS), free radical nitrile (NO), anti-inflammatory activity and GSH depletion. The conjugates showed significant increase in antioxidant activity (more than a factor of 2) compared to free drug as seen from the inhibition of LPS induced ROS, NO, GSH and tumor necrosis factor-alpha (TNF-alpha) release in microglial cells.

Anti-inflammatory and anti-oxidant activity of anionic dendrimer-N-acetyl cysteine conjugates in activated microglial cells.

Dendrimers are emerging as potential intracellular drug delivery vehicles. Understanding and improving the cellular efficacy of dendrimer-drug conjugates, can lead to significant in vivo benefits. This study explores efficacy of anionic polyamidoamine (PAMAM-COOH) dendrimer-N-acetyl cysteine (NAC) conjugates for applications in neuroinflammation. The anti-oxidative and anti-inflammatory effects of PAMAM-(COOH)(46)-(NAC)(18) conjugate is evaluated on microglial cells in vitro. Cell entry and localization of PAMAM-(COOH)(62)-(FITC)(2) conjugate in BV-2 microglial cells were assessed using flow cytometry and confocal microscopy. ELISA assays were used to evaluate markers of oxidative stress (ROS, NO) and inflammation (TNF-alpha) after stimulation of microglial cells with lipopolysaccharides (LPS), following treatment with increasing doses of free N-acetyl-L-cysteine (NAC) or PAMAM-(COOH)(46)-(NAC)(18) conjugate containing an equivalent molar concentration of NAC. Flow cytometry and confocal microscopy demonstrated the PAMAM-(COOH)(62)-(FITC)(2) conjugate entered BV-2 cells rapidly with significant increase in fluorescence within 15 min and localized mostly in the cytoplasm. PAMAM-(COOH)(46)-(NAC)(18) conjugate was non-toxic, and significantly reduced ROS, NO and TNF-alpha release by activated microglial cells after 24 h and 72 h stimulation of LPS following 3h pre-treatment when compared to the same concentration of free NAC (P<0.05 or P<0.01). Anionic PAMAM dendrimer-NAC conjugate was synthesized with a glutathione sensitive linker for intracellular release. The non-toxic conjugate is a more effective anti-oxidant and anti-inflammatory agent when compared to free NAC in vitro. The conjugate showed significant efficacy even at the lowest dose (0.5mM NAC), where the activity was comparable or better than that of free drug at 8mM (16x higher dosage). The improved efficacy of the conjugate, when combined with the intrinsic neuroinflammation-targeting ability of the PAMAM dendrimers, may provide new opportunities for in vivo applications.

Poly(amidoamine) dendrimer-drug conjugates with disulfide linkages for intracellular drug delivery.

Understanding and improving drug release kinetics from dendrimer-drug conjugates are key steps to improve their in vivo efficacy. N-Acetyl cysteine (NAC) is an anti-inflammatory agent with significant potential for clinical use in the treatment of neuroinflammation, stroke and cerebral palsy. There is a need for delivery of NAC which can enhance its efficacy, reduce dosage and prevent it from binding plasma proteins. For this purpose, a poly(amidoamine) dendrimer-NAC conjugate that contains a disulfide linkage was synthesized and evaluated for its release kinetics in the presence of glutathione (GSH), cysteine (Cys), and bovine serum albumin (BSA) at both physiological and lysosomal pH. The results indicate that the prepared conjugate can deliver approximately 60% of its NAC payload within 1h at intracellular GSH concentrations at physiological pH, whereas the conjugate did not release any drug at plasma GSH levels. The stability of the conjugate in the presence of bovine serum albumin at plasma concentrations was also demonstrated. The efficacy of the dendrimer-NAC conjugate was measured in activated microglial cells (target cells in vivo) using the reactive oxygen species (ROS) assay. The conjugates showed an order of magnitude increase in antioxidant activity compared to free drug. When combined with intrinsic and ligand-based targeting with dendrimers, these types of GSH sensitive nanodevices can lead to improved drug release profiles and in vivo efficacy.

Dendrimer-drug conjugates for tailored intracellular drug release based on glutathione levels.

N-Acetyl-L-cysteine (NAC) is an antioxidant and anti-inflammatory agent with significant potential in clinical applications including stroke and neuroinflammation. The drug shows high plasma binding upon IV administration, requiring high doses and associated side effects. Through the use of an appropriate delivery vehicle, the stability and efficacy of NAC can be significantly improved. Dendrimers are an emerging class of nanoscale drug delivery vehicles, which enable high drug payloads and intracellular delivery. Poly(amidoamine) (PAMAM) dendrimer-NAC conjugates having cleavable disulfide linkages are designed for intracellular delivery based on glutathione levels. We have successfully synthesized two conjugates with a cationic G4-NH(2) and an anionic G3.5-COOH PAMAM dendrimer with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by (1)H NMR and MALDI-TOF analysis. NAC release from the conjugates at intracellular and extracellular glutathione (GSH) concentrations were evaluated by reverse phase HPLC (RP-HPLC) analysis, and approximately 70% of NAC payload was released within one hour at intracellular GSH concentrations (approximately 10 mM), whereas negligible NAC release was observed at extracellular GSH levels (2 microM). FITC-labeled conjugates showed that they enter cells rapidly and localize in the cytoplasm of lipopolysaccharide (LPS)-activated microglial cells (the target cells in vivo). The significantly improved efficacies of dendrimer-NAC conjugates in activated microglial cells was confirmed by measuring the nitrite inhibition in the cell culture medium, which is an indication of the antioxidative property of the drug. Both G4-NH(2) and G3.5-COOH conjugates showed significantly better nitrite inhibition both at 24 and 72 h compared to free NAC, by as much as a factor of 16. The results indicate that PAMAM dendrimer conjugates produce higher local NAC concentration inside the cells, with GSH-sensitive disulfide linker enabling efficient and rapid cellular release of the drug.