Sujatha Kannan

Drug complexation, in vitro release and cellular entry of dendrimers and hyperbranched polymers

Highly branched, functionalized polymers have potential to act as efficient drug carrier systems. Dendrimers are ideal candidates among model hyperbranched polymers because of their well-defined structure and high density of functional groups. Using ibuprofen as a model drug, we studied the interaction between the drug and Polyamidoamine (PAMAM) dendrimers (generations 3 and 4 with --NH2 functionality) and Perstrop Polyol (generation 5, hyperbranched polyester with --OH functionality). FTIR and NMR studies suggest that ibuprofen predominantly forms a complex with PAMAM dendrimers because of the ionic interaction between the --NH2 end groups and the carboxyl group of ibuprofen. On an average, up to 78 molecules of ibuprofen could be incorporated into one molecule of PAMAM-G4-NH2 with 64 end groups. This complex is stable in deionized water and methanol. The in vitro release of ibuprofen from drug-dendrimer complex is appreciably slower compared to pure ibuprofen. The complexed drug enters A549 cells much more rapidly than pure drug suggesting that dendrimers may be able to carry the complexed drug inside cells efficiently. Hyperbranched Polyol (with 128 --OH end groups) appears to encapsulate approximately 24 drug molecules. Perhaps the lack of strong interactions between the --OH end groups and the drugs prevents complex formation.

The effect of surface functionality on cellular trafficking of dendrimers

Dendrimers are an emerging group of nanostructured, polymeric biomaterials that have potential as non-viral vehicles for delivering drugs and genetic material to intracellular targets. They have a high charge density with tunable surface functional groups, which can alter the local environment and influence cellular interactions. This can have a significant impact on the intracellular trafficking of dendrimer-based nanodevices. With the help of flow cytometry, fluorescence microscopy, and by using specific inhibitors, the influence of surface functionality on their uptake in A549 lung epithelial cells, and subsequent intracellular distribution was investigated. In this paper, we have shown that even though all the dendrimers are taken up by fluid-phase endocytosis, significant differences in uptake mechanisms exist. Anionic dendrimers appear to be mainly taken up by caveolae mediated endocytosis in A549 lung epithelial cells, while cationic and neutral dendrimers appear to be taken in by a non-clathrin, non-caveolae mediated mechanism that may be by electrostatic interactions or other non-specific fluid-phase endocytosis. These findings open up new possibilities of targeting therapeutic agents to specific cell organelles based on surface charge.

Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications

Dendrimers are discrete nanostructures/nanoparticles with ‘onion skin‐like’ branched layers. Beginning with a core, these nanostructures grow in concentric layers to produce stepwise increases in size that are similar to the dimensions of many in vivo globular proteins. These branched tree‐like concentric layers are referred to as ‘generations’. The outer generation of each dendrimer presents a precise number of functional groups that may act as a monodispersed platform for engineering favourable nanoparticle–drug and nanoparticle–tissue interactions. These features have attracted significant attention in medicine as nanocarriers for traditional small drugs, proteins, DNA/RNA and in some instances as intrinsically active nanoscale drugs. Dendrimer‐based drugs, as well as diagnostic and imaging agents, are emerging as promising candidates for many nanomedicine applications. First, we will provide a brief survey of recent nanomedicines that are either approved or in the clinical approval process. This will be followed by an introduction to a new ‘nanoperiodic’ concept which proposes nanoparticle structure control and the engineering of ‘critical nanoscale design parameters’ (CNDPs) as a strategy for optimizing pharmocokinetics, pharmocodynamics and site‐specific targeting of disease. This paradigm has led to the emergence of CNDP‐directed nanoperiodic property patterns relating nanoparticle behaviour to critical in vivo clinical translation issues such as cellular uptake, transport, elimination, biodistribution, accumulation and nanotoxicology. With a focus on dendrimers, these CNDP‐directed nanoperiodic patterns are used as a strategy for designing and optimizing nanoparticles for a variety of drug delivery and imaging applications, including a recent dendrimer‐based theranostic nanodevice for imaging and treating cancer. Several emerging preclinical dendrimer‐based nanotherapy concepts related to inflammation, neuro‐inflammatory disorders, oncology and infectious and ocular diseases are reviewed. Finally we will consider challenges and opportunities anticipated for future clinical translation, nanotoxicology and the commercialization of nanomedicine.

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

A dendrimer-drug conjugate attenuates neuroinflammation and improves motor function in a rabbit model of cerebral palsy. One Hop at a Time Cerebral palsy (CP) is a developmental disorder caused by injury to a baby’s brain while it is still developing, either in the womb or during the early months of life, but is often not diagnosed until children are 2 to 3 years of age. There is no cure for CP, and the best option for affected children is intensive physical therapy to improve motor skills. Now, Kannan et al. have designed a dendrimer-based therapeutic for treating this developmental disorder in baby rabbits (kits), opening the door to new treatment options in humans. The authors chose to use the rabbit model of CP, which replicates the neuroinflammation seen in human brains as well as the motor deficits in children. To generate this model, Kannan and colleagues injected Escherichia coli toxin into the rabbit mother’s uterus at about 90% term gestation. When the kits were born, they were administered either a saline solution, a free drug known as NAC (N-acetyl-l-cysteine), or a dendrimer-NAC (D-NAC) conjugate. This postnatal “rescue” with D-NAC, given on day 1 of life, allowed CP kits to develop normally, able to walk and hop. The successfully treated kits also had neuron counts and low inflammation similar to healthy control animals. By comparison, NAC alone or saline had no effect. The authors believe that conjugating NAC to the dendrimers promoted greater uptake by activated microglia and astrocytes, with no toxicity to surrounding neurons. Although still in preclinical testing in rabbits, this dendrimer-drug conjugate shows promise for postnatal treatment of babies suspected of having CP. 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.

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.

Models of Fetal Brain Injury, Intrauterine Inflammation, and Preterm Birth

Intrauterine infection and inflammation are known risk factors for brain damage in the neonate irrespective of the gestational age. Infection‐induced maternal immune activation leads to a fetal inflammatory response mediated by cytokines that has been implicated in the development of not only periventricular leukomalacia and cerebral palsy but also a spectrum of neurodevelopmental disorders such as autism and schizophrenia (Behav Brain Res 2009; 204:313, Ann Neurol 2005; 57:67, Am J Obstet Gynecol 2000; 182:675). A common link among the neurobehavioral disorders associated with intrauterine inflammation appears to be the evidence for immune dysregulation in the developing brain (Behav Brain Res 2009; 204:313). The timing of the immune challenge with respect to the gestational age and neurologic development of the fetus may be crucial in the elicited response (J Neurosci 2006; 26:4752). Studies involving animal models of maternal inflammation serve a key role in elucidation of mechanisms involved in fetal brain injury associated with exposure to the maternal milieu. These animal models have been shown to result in fetal microglial activation, neurotoxicity as well motor deficits and behavioral abnormalities in the offspring (J Neurosci 2006; 26:4752, J Neurosci Res 2010; 88:172, Am J Obstet Gynecol 2009; 201:279, Am J Obstet Gynecol 2008; 199:651). A better understanding of the mechanisms of perinatal brain injury will allow discoveries of novel neuroprotective agents, better outcomes following preterm birth and stratification of fetuses and neonates for therapies in cases of preterm birth, preterm premature rupture of membranes, and chorioamnionitis.

Intrauterine administration of endotoxin leads to motor deficits in a rabbit model: a link between prenatal infection and cerebral palsy

OBJECTIVE This study was undertaken to determine whether maternal intrauterine endotoxin administration leads to neurobehavioral deficits in newborn rabbits. STUDY DESIGN Pregnant New Zealand white rabbits were injected with 1 mL saline solution (n = 8) or 20 microg/kg of lipopolysaccharide in saline solution (n = 8) into the uterine wall on day 28/31 of gestation. On postnatal day 1, kits (saline solution [n = 30] and lipolysaccharide in saline solution [n = 18] from 4 consecutive litters) underwent neurobehavioral testing. Neonatal brains were stained for microglial cells and myelin. RESULTS Kits in the lipopolysaccharide in saline solution group were hypertonic and demonstrated significant impairment in posture, righting reflex, locomotion, and feeding, along with neuroinflammation indicated by activated microglia and hypomyelination in the periventricular regions. A greater mortality was noted in the lipopolysaccharide in saline solution group (16 stillbirths from 3 litters vs 3 from 1 litter). CONCLUSION Maternal intrauterine endotoxin administration leads to white matter injury and motor deficits in the newborn rabbit, resulting in a phenotype that resembles those found in periventricular leukomalacia and cerebral palsy.

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, Alzheimers and multiple sclerosis.

Microglial Activation in Perinatal Rabbit Brain Induced by Intrauterine Inflammation: Detection with 11C-(R)-PK11195 and Small-Animal PET

Intrauterine infection can lead to a fetal inflammatory response syndrome that has been implicated as one of the causes of perinatal brain injury leading to periventricular leukomalacia (PVL) and cerebral palsy. The presence of activated microglial cells has been noted in autopsy specimens of patients with PVL and in models of neonatal hypoxia and ischemia. Activated microglial cells can cause oligodendrocyte damage and white matter injury by release of inflammatory cytokines and production of excitotoxic metabolites. We hypothesized that exposure to endotoxin in utero leads to microglial activation in the fetal brain that can be monitored in vivo by 11C-(R)-PK11195 (1-[2-chlorophenyl]-N-methyl-N-[1-methylpropyl]-3-isoquinoline carboxamide)—a positron-emitting ligand that binds peripheral benzodiazepine receptor sites in activated microglia—using small-animal PET. Methods: Pregnant New Zealand White rabbits underwent laparotomy and were injected with 20 and 30 μg/kg of Escherichia coli lipopolysaccharide along the length of the uterus on day 28 of gestation. The pups were born spontaneously at term (31 d) and were scanned using small-animal PET after intravenous administration of 11C-(R)-PK11195 and by MRI on postnatal day 1. The standard uptake values (SUVs) of the tracer were calculated for the whole brain at 10-min intervals for 60 min after tracer injection. The pups were euthanized after the scan, and brains were fixed, sectioned, and stained for microglial cells using biotinylated tomato lectin. Results: There was increased brain retention of 11C-(R)-PK11195—as determined by a significant difference in the slope of the SUV over time—in the endotoxin-treated pups when compared with that of age-matched controls. Immunohistochemical staining showed dose-dependent changes in activated microglia (increased number and morphologic changes) in the periventricular region and hippocampus of the brain of newborn rabbit pups exposed to endotoxin in utero. Conclusion: Intrauterine inflammation leads to activation of microglial cells that may be responsible for the development of brain injury and white matter damage in the perinatal period. PET with the tracer 11C-(R)-PK11195 can be used as a noninvasive, sensitive tool for determining the presence and progress of neuroinflammation due to perinatal insults in newborns.

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.