Janie Baratta

Effective Targeting of Liposomes to Liver and Hepatocytes In Vivo by Incorporation of a Plasmodium Amino Acid Sequence

PurposeSeveral species of the protozoan Plasmodium effectively target mammalian liver during the initial phase of host invasion. The purpose of this study was to demonstrate that a Plasmodium targeting amino acid sequence can be engineered into therapeutic nanoparticle delivery systems.MethodsA 19-amino peptide from the circumsporozoite protein of Plasmodium berghei was prepared containing the conserved region I as well as a consensus heparan sulfate proteoglycan binding sequence. This peptide was attached to the distal end of a lipid–polyethylene glycol bioconjugate. The bioconjugate was incorporated into phosphatidylcholine liposomes containing fluorescently labeled lipids to follow blood clearance and organ distribution in vivo.ResultsWhen administered intravenously into mice, the peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver. Fluorescence and electron microscopy demonstrated that the liposomes were accumulated both by nonparenchymal cells and hepatocytes, with the majority of the liposomal material associated with hepatocytes. Accumulation of liposomes in the liver was several hundredfold higher compared to heart, lung, and kidney, and more than 10-fold higher compared to spleen. In liver slice experiments, liposome binding was specific to sites sensitive to heparinase.ConclusionsIncorporation of amino acid sequences that recognize glycosaminoglycans is an effective strategy for the development of targeted drug delivery systems.

Do subplate neurons comprise a transient population of cells in developing neocortex of rats

Studies were undertaken to determine whether neurons of the subplate layer represent a transient or stable population of cells in developing neocortex of rat. The first set of studies sought to determine the fraction of subplate neurons that is lost during early postnatal development. The optical dissector method was used to analyze fluorescently stained material in animals the age of postnatal day 0 (P0) to P40. These results demonstrate a reduction of slightly less than half of the total number of subplate neurons from P0 to P40. Counts of labeled cells in littermates at varied ages after [3H]thymidine or BRDU treatment on gestational day 14 (G14 ‐ birthdate of occipital subplate neurons) or G18 (birthdate of layers III–IV neurons) demonstrate loss of approximately 50% of neurons in the subplate layer between P0 and P40, somewhat greater than the loss of neurons from cortical layers III–IV. The second set of studies investigated whether subplate neurons display cellular atrophy during postnatal development. Analysis of subplate neurons injected intracellularly with Lucifer yellow in fixed slice preparations indicates no reduction in soma size, number of dendrites, or extent of dendritic fields of subplate neurons taken from animals age P0 to P60. The third set of studies investigated whether functional markers of subplate neurons are reduced during postnatal development. Analysis of tissue stained histochemically for cytochrome oxidase or acetylcholinesterase, or stained immunocytochemically for GABA, somatostatin, or neuropeptide Y, demonstrate a remarkable loss of expression of staining patterns from late gestational ages to P20. These data demonstrate that, although subplate neurons seem not to be a transient population of cells in the usual sense of being eliminated by cell death or structural atrophy, the loss of histochemical and immunocytochemical markers indicates that they may be a functionally transient population of cells. J. Comp. Neurol. 426:632–650, 2000.

AFGF promotes axonal growth in rat spinal cord organotypic slice co-cultures.

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15-P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.

Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors.

Axon growth failure in the central nervous system (CNS) of adult animals is thought to be attributable to several factors, including an inadequate intrinsic growth response, the presence of inhibitory molecules, and a lack of adequate neurotrophic support. Here we use a new in vitro assay system to quantitatively assess growth of axons in cortex/spinal cord organotypic co-cultures from neonatal rats. Co-cultures of cortex and spinal cord were prepared from neonatal rats at P3 or P7, and by pairing cortex and spinal cords from different ages. Axon growth from the cortex to the spinal cord was assessed using DiI tract tracing techniques. Axons could be traced from the cortex to the spinal cord in co-cultures in which both tissues were obtained from P3 animals, whereas few axons crossed the cortex/spinal cord boundary in co-cultures from P7 animals. A larger number of axons could be traced across the boundary in co-cultures from P3 animals that were treated with neurotrophins (NGF, BDNF, or NT3), whereas neurotrophins produced minimal growth enhancement in P7 co-cultures. In mixed age co-cultures of P7 cortex with P3 spinal cord, moderate numbers of axons extended between the cortex and spinal cord when cultures were treated with neurotrophins, but few if any crossing axons were detected in co-cultures of P3 cortex with P7 spinal cords. These results indicate that successful growth of axons from the cortex to the spinal cord depends on the developmental age of the tissue terrain (the spinal cord and/or the interface between cortex and spinal cord explants), and to a lesser extent on the developmental state of the cortical neurons, and that axon growth between cortex and spinal cord can be enhanced by exogenous neurotrophins. These co-cultures provide a potentially useful assay for factors that affect axon growth that is intermediate between assays based on dissociated neurons and the intact tissue terrain.

Liposomal delivery of doxorubicin to hepatocytes in vivo by targeting heparan sulfate

Previous work demonstrated that liposomes, containing an amino acid sequence that binds to hepatic heparan sulfate glycosaminoglycan, show effective targeting to liver hepatocytes. These liposomes were tested to determine whether they can deliver doxorubicin selectively to liver and hepatocytes in vivo. Fluid-phase liposomes contained a lipid-anchored 19-amino acid glycosaminoglycan targeting peptide. Liposomes were loaded with doxorubicin and were non-leaky in the presence of serum. After intravenous administration to mice, organs were harvested and the doxorubicin content extracted and measured by fluorescence intensity and by fluorescence microscopy. The liposomal doxorubicin was recovered almost entirely from liver, with only trace amounts detectable in heart, lung, and kidney. Fluorescence microscopy demonstrated doxorubicin preferentially in hepatocytes, also in non-parenchymal cells of the liver, but not in cells of heart, lung or kidney. The doxorubicin was localized within liver cell nuclei within 5 min after intravenous injection. These studies demonstrated that liposomal doxorubicin can be effectively delivered to hepatocytes by targeting the heparan sulfate glycosaminoglycan of liver tissue. With the composition described here, the doxorubicin was rapidly released from the liposomes without the need for an externally supplied stimulus.

Absence of selectivity in the loss of neurons from the developing cortical subplate of the rat

Neurons of the cortical subplate display evidence of cell death, although a significant population survives to the mature brain. The present study examined different populations of neurons to determine if the loss of cells was specific for a particular cell type. Immunocytochemical procedures for neurons expressing GluR2/3, GAD, or NPY, were used on tissue sections taken from animals at gestational day 18 to postnatal day 21. The rate of loss of labeled cells was similar for all groups of neurons. Thus, these data reveal no evidence that the loss of subplate neurons is specific to any major cell type.

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus.

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells. These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.

A role for endocannabinoids in viral-induced dyskinetic and convulsive phenomena

Dyskinesias and seizures are both medically refractory disorders for which cannabinoid-based treatments have shown early promise as primary or adjunctive therapy. Using the Borna disease (BD) virus rat, an animal model of viral encephalopathy with spontaneous hyperkinetic movements and seizure susceptibility, we identified a key role for endocannabinoids in the maintenance of a balanced tone of activity in extrapyramidal and limbic circuits. BD rats showed significant elevations of the endocannabinoid anandamide in subthalamic nucleus, a relay nucleus compromised in hyperkinetic disorders. While direct and indirect cannabinoid agonists had limited motor effects in BD rats, abrupt reductions of endocannabinoid tone by the CB1 antagonist SR141716A (0.3 mg/kg, i.p.) caused seizures characterized by myoclonic jerks time-locked to periodic spike/sharp wave discharges on hippocampal electroencephalography. The general opiate antagonist naloxone (NLX) (1 mg/kg, s.c.), another pharmacologic treatment with potential efficacy in dyskinesias or L-DOPA motor complications, produced similar seizures. No changes in anandamide levels in hippocampus and amygdala were found in convulsing NLX-treated BD rats. In contrast, NLX significantly increased anandamide levels in the same areas of normal uninfected animals, possibly protecting against seizures. Pretreatment with the anandamide transport blocker AM404 (20 mg/kg, i.p.) prevented NLX-induced seizures. These findings are consistent with an anticonvulsant role for endocannabinoids, counteracting aberrant firing produced by convulsive agents, and with a functional or reciprocal relation between opioid and cannabinoid tone with respect to limbic convulsive phenomena.

Cholinergic innervation of cerebral cortex in organotypic slice cultures: Sustained basal forebrain and transient striatal cholinergic projections

Slices of entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic slice cultures. Basal forebrain cholinergic neurons, identified by histochemical staining for acetylcholinesterase, develop axons that grow rapidly into cerebral cortex. Ingrowth occurs by two routes: some axons course laterally from the basal forebrain region to reach lateral neocortex; others course dorsally from the septum to reach medial cortex. By one to two weeks in vitro, acetylcholinesterase-positive axons have extended throughout most of the cortical territory. In addition to basal forebrain cholinergic axons, the normally local circuit cholinergic neurons of the striatum also send axons into cerebral cortex. These striatum-derived axons can be distinguished from basal forebrain axons by their distinct morphological characteristics and by their different response to excision of the striatum or basal forebrain. Further, acetylcholinesterase-positive axons in cortex that originate from striatum appear to retract or degenerate after about one week in culture, while those from basal forebrain remain present and apparently healthy beyond two weeks. These data document the basal forebrain cholinergic ingrowth into cerebral cortex using this whole hemisphere slice culture system and also demonstrate different degrees of maintenance of cortical afferents that are derived from different subcortical sources.

Liposomal polyethyleneglycol and polyethyleneglycol-peptide combinations for active targeting to liver in vivo.

This report describes the development and evaluation of a range of polyethyleneglycol and polyethyleneglycol-peptide liposome formulations that effectively target liver in vivo. A 19-amino-acid sequence from the N-terminal region of the circumsporozoite protein of Plasmodium berghei was attached to the distal end of di22:1-aminopropane-polyethyleneglycol3400, and incorporated into liposomes containing di22:1-phosphatidylcholine and di22:1-phosphatidylethanolamine-polyethyleneglycol5000. By systematically varying the mole fractions of both the lipid-polyethyleneglycol and the lipid-polyethyleneglycol-peptide conjugates, and screening for serum-induced aggregation in vitro, a serum-stable range of formulations was established. These stable formulations were tested for binding to Hepa 1-6 liver cells in culture, and from these results three formulations were prepared for intravenous administration in mice. All three formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min. Uptake by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Effective targeting to liver in vivo was successful after repeated (up to three) administrations to the host at 14-day intervals. All formulations prepared for in vivo administration were stable in the presence of serum, as measured by complete retention of entrapped calcein dye. The formulation with the lowest mole fractions of peptide and polyethyleneglycol was the most cost-effective in terms of encapsulation efficiency and minimal use of peptide and polymer compounds. The in vitro biophysical screening, followed by cell culture testing, reduced the number of animals required to develop an effective set of targeted liposome formulations for in vivo application.