Kinetic Properties and Pharmacological Modulation of High- and Low-Affinity Dopamine Transport in Striatal Astrocytes of Adult Rats.

Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [3H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [3H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders.

Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake.

Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.

Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

Cardiopulmonary Effects and Pharmacokinetics of Dexmedetomidine Used as an Adjunctive Analgesic to Regional Anesthesia of the Oral Cavity with Levobupivacaine in Dogs.

This study investigated the cardiopulmonary effects and pharmacokinetics of dexmedetomidine (DEX) used as an adjunctive analgesic for regional anesthesia of the oral cavity with levobupivacaine in anesthetized dogs. Forty dogs were randomly assigned to four groups of 10 dogs. All dogs received levobupivacaine (4 blocks) with DEX IO (infraorbital block, n = 10) or IA (inferior alveolar block, n = 10) or placebo (PLC; n = 10) or DEX (n = 10) was injected intravenously (IV) after administration of levobupivacaine. The dose of DEX was always 0.5 µg/kg. Cardiopulmonary parameters were recorded, and blood was drawn for the quantification of DEX in plasma using LC-MS/MS. Heart rate was lower in all LB + DEX groups, while mean arterial pressure (MAP) was higher in the LB + DEX IV and LB + DEX IA groups compared to the LB + PLC IV group. Compared to DEX IV, IO and IA administration resulted in lower MAP up to 2 min after application. Absorption of DEX was faster at IO administration (Cmax and Tmax were 0.47 ± 0.08 ng/mL and 7.22 ± 1.28 min and 0.76 ± 0.09 ng/mL and 7.50 ± 1.63 min for the IO and IA block, respectively). The IA administration resulted in better bioavailability and faster elimination (t1/2 was 63.44 ± 24.15 min and 23.78 ± 3.78 min for the IO and IA block, respectively). Perineural administration of DEX may be preferable because of the less pronounced cardiovascular response compared to IV administration.

The pharmacokinetics of levobupivacaine 0.5% after infraorbital or inferior alveolar block in anesthetized dogs.

Introduction: Data are lacking on the pharmacokinetic profile and safety of levobupivacaine (LB) used for regional anesthesia of the maxilla and mandibles in dogs. Methods: Infraorbital block (n = 10), inferior alveolar block (n = 10) or both infraorbital and inferior alveolar blocks (n = 10) were administered to dogs undergoing dental surgery under isoflurane anesthesia. The dose of LB was calculated as 0.11 ml/kg2/3 for the infraorbital block and 0.18 ml/kg2/3 for the inferior alveolar block. Blood samples were collected before and immediately after administration of the oral blocks, and 3, 4, 7, 12, 17, 32, 47, 62, 92, and 122 min thereafter. Quantification of LB in plasma was performed by LC-MS/MS. Results and discussion: The results are presented as median and interquartile range. In dogs in which all four quadrants of the oral cavity were desensitized with LB, the C max was 1,335 (1,030-1,929) ng/ml, the T max was 7 (4-9.5) min, and the AUC(0 → 120) was 57,976 (44,954-96,224) ng min/ml. Plasma concentrations of LB were several times lower than the reported toxic concentrations, and no signs of cardiovascular depression or neurotoxicity were observed in any of the dogs, suggesting that the occurrence of severe adverse effects after administration of LB at the doses used in this study is unlikely.

Bleomycin Concentration in Patients' Plasma and Tumors after Electrochemotherapy. A Study from InspECT Group.

The plasma concentration profile of bleomycin in the distribution phase of patients younger than 65 years is needed to determine the suitable time interval for efficient application of electric pulses during electrochemotherapy. Additionally, bleomycin concentrations in the treated tumors for effective tumor response are not known. In this study, the pharmacokinetic profile of bleomycin in the distribution phase in 12 patients younger than 65 years was determined. In 17 patients, the intratumoral bleomycin concentration was determined before the application of electric pulses. In younger patients, the pharmacokinetics of intravenously injected bleomycin demonstrated a faster plasma clearance rate than that in patients older than 65 years. This outcome might indicate that the lowering of the standard bleomycin dose of 15,000 IU/m2 with intravenous bleomycin injection for electrochemotherapy is not recommended in younger patients. Based on the plasma concentration data gathered, a time interval for electrochemotherapy of 5-15 min after bleomycin injection was determined. The median bleomycin concentration in tumors 8 min after bleomycin injection, at the time of electroporation, was 170 ng/g. Based on collected data, the reduction of the bleomycin dose is not recommended in younger patients; however, a shortened time interval for application of electric pulses in electrochemotherapy to 5-15 min after intravenous bleomycin injection should be considered.

Pharmacokinetics of carprofen in anaesthetized pigs: a preliminary study.

OBJECTIVE: To investigate the pharmacokinetics of carprofen after a single intravenous (IV) dose and multiple oral doses administered to pigs undergoing electroporation of the pancreas. STUDY DESIGN: Prospective experimental study. ANIMALS: A group of eight female pigs weighing 31.74 ± 2.24 kg (mean ± standard deviation). METHODS: Carprofen 4 mg kg-1 was administered IV after placement of a central venous catheter during general anaesthesia with isoflurane. Blood samples were collected 30 seconds before and 5, 10, 20, 30 and 60 minutes and 2, 4, 6, 8, 12 and 24 hours after carprofen administration. Subsequently, the same dose of carprofen was administered orally, daily, for 6 consecutive days and blood collected at 36, 48, 60, 72, 96, 120, 144 and 168 hours after initial carprofen administration. Plasma was analysed using liquid chromatography with mass spectrometry. Standard pharmacokinetic parameters were calculated by compartmental analysis of plasma concentration-time curves. Data are presented as mean ± standard error. RESULTS: The initial plasma concentration of IV carprofen was estimated at 54.57 ± 3.92 µg mL-1 and decreased to 8.26 ± 1.07 µg mL-1 24 hours later. The plasma elimination curve showed a bi-exponential decline: a rapid distribution phase with a distribution half-life of 0.21 ± 0.03 hours and a slower elimination phase with an elimination half-life of 17.31 ± 3.78 hours. The calculated pharmacokinetic parameters were as follows: the area under the plasma concentration-time curve was 357.3 ± 16.73 µg mL-1 hour, volume of distribution was 0.28 ± 0.07 L kg-1 and plasma clearance rate was 0.19 ± 0.009 mL minute-1 kg-1. The plasma concentration of carprofen, administered orally from days 2 to 7, varied from 9.03 ± 1.87 to 11.49 ± 2.15 µg mL-1. CONCLUSIONS AND CLINICAL RELEVANCE: Carprofen can be regarded as a long-acting non-steroidal anti-inflammatory drug in pigs.

Case Report: Intoxication in a Pig (Sus Scrofa Domesticus) After Transdermal Fentanyl Patch Ingestion.

An experimental study on the effects of electroporation on pancreatic tissue was performed in pigs, and the fentanyl transdermal patch (FTP) was used postoperatively as part of multimodal pain management. Ingestion of an FTP, which resulted in fentanyl intoxication, was suspected 5 days after placement in one of the experimental pigs. The pig was first dysphoric, running in the stall, panting and vocalizing until it finally became depressed and it remained lying on the floor. Ingestion of an FTP was not observed but the fentanyl plasma concentration on the day of intoxication was 20.7 ng/ml, while at its peak after FTP administration it was only 0.492 ng/ml. The intoxication was successfully treated with a single intramuscular naloxone injection.

Relevance of Hydrogen Bonds for the Histamine H2 Receptor-Ligand Interactions: A Lesson from Deuteration.

We used a combination of density functional theory (DFT) calculations and the implicit quantization of the acidic N-H and O-H bonds to assess the effect of deuteration on the binding of agonists (2-methylhistamine and 4-methylhistamine) and antagonists (cimetidine and famotidine) to the histamine H2 receptor. The results show that deuteration significantly increases the affinity for 4-methylhistamine and reduces it for 2-methylhistamine, while leaving it unchanged for both antagonists, which is found in excellent agreement with experiments. The revealed trends are interpreted in the light of the altered strength of the hydrogen bonding upon deuteration, known as the Ubbelohde effect, which affects ligand interactions with both active sites residues and solvent molecules preceding the binding, thus providing strong evidence for the relevance of hydrogen bonding for this process. In addition, computations further underline an important role of the Tyr250 residue for the binding. The obtained insight is relevant for the therapy in the context of (per)deuterated drugs that are expected to enter therapeutic practice in the near future, while this approach may contribute towards understanding receptor activation and its discrimination between agonists and antagonists.

Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy.

Pre-clinical and clinical data indicate differences in the responses of melanoma and carcinoma tumours to electrochemotherapy. The purpose of this study was to investigate the origin of this difference, whether it is due to the intrinsic difference in tumour cell susceptibility to the chemotherapeutic, or due to the tumour micro-environment. For this purpose, we performed a pre-clinical study in B16F1 melanoma and TS/A carcinoma tumours in mice, in which the antitumour effectiveness of electrochemotherapy with bleomycin, the intrinsic sensitivity of tumour cells in vitro, the pharmacokinetics of bleomycin in plasma and tumours, and the vascularization of tumours in vivo were evaluated. The results of the treatment show that carcinoma was significantly more responsive to electrochemotherapy than melanoma. This effect cannot be ascribed to the intrinsic sensitivity of these cells, as melanoma cells were more sensitive than carcinoma cells in vitro. The difference in responses could be ascribed to differences in the pharmacokinetics of bleomycin; at the time of electroporation in carcinomas, more bleomycin was accumulated. This effect could be due to differences in tumour vascularization, as carcinoma tumours had numerous well-distributed, small blood vessels, while melanomas were less vascularized, exhibiting predominantly larger vessels. In conclusion, this study provides evidence on the importance of the tumour micro-environment, particularly the tumour vasculature, in the responses of the tumours to bleomycin electrochemotherapy. Vasculature is important for the pharmacokinetics of bleomycin, influencing drug accumulation and drug distribution in tumours, and might be used as a predictive factor for the tumour response to electrochemotherapy.

Efficiency of electrochemotherapy with reduced bleomycin dose in the treatment of nonmelanoma head and neck skin cancer: Preliminary results.

BACKGROUND: In the present study, effectiveness of electrochemotherapy was compared in patients with nonmelanoma skin cancer (NMSC) of the head and neck using standard and reduced doses of bleomycin. METHODS: Twenty-eight patients older than 65 years were prospectively treated with electrochemotherapy for nonmelanoma head and neck skin cancer. In the experimental group (n = 12 patients; 24 lesions), reduced bleomycin doses (10 000 IU/m2 ) were used. In the control group (n = 16 patients; 28 lesions), the standard bleomycin doses (15 000 IU/m2 ) were used. Tumor responses and side effects were monitored. These two groups were pair matched for the characteristics of patients (age, gender) and their tumors (diameter, histology type, recurrent lesions). RESULTS: Complete tumor response at 2 months post-electrochemotherapy with the reduced bleomycin dose was 100% and with the standard bleomycin dose it was 96%. No statistically significant difference regarding skin toxicity was observed between the 2 groups (P = .20). Skin toxicity of grade 3 or less was recorded only in the control group (7% of treated lesions). CONCLUSION: Presented results show the comparable antitumor effectiveness of electrochemotherapy when using standard or reduced bleomycin dose in elderly patients with nonmelanoma head and neck skin cancer.

Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome.

Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

Histamine and astrocyte function.

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure.

Bleomycin pharmacokinetics of bolus bleomycin dose in elderly cancer patients treated with electrochemotherapy.

PURPOSE: With the aim to determine effective therapeutic window of electrochemotherapy, we analyzed bleomycin pharmacokinetic parameters in elderly patients. METHODS: In prospective clinical study in the treatment of tumors with electrochemotherapy, blood samples of patients older than 65 years were collected after the bolus intravenous injection of bleomycin (15,000 IU/m(2)). In serum samples, quantitative analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. Based on the data, the pharmacokinetic parameters of bleomycin elimination were determined. RESULTS: Pharmacokinetic analysis of the data revealed a monophasic serum clearance curve, which demonstrates slow elimination of bleomycin, being less than 500 ml/min and a half-time of 30 min. CONCLUSIONS: Slow monophasic elimination of bleomycin from serum in elderly patients implies on the longer therapeutic window, from 8 to up to 40 min or even longer post-bleomycin injection for electrochemotherapy. However, prolonged therapeutic bleomycin serum concentrations may also affect the possible adverse effects, such as lung fibrosis and extensive necrosis of tumors due to the uptake of toxic bleomycin concentrations into the tumors. This may imply on lowering of bleomycin dosage, in particular in the elderly patients.

The prevalence and pattern of pharmaceutical and excipient exposure in a neonatal unit in Slovenia.

OBJECTIVE: Because of the restraints on conducting studies on pharmaceutical use in sick newborns, many drugs are used off-label in this population. Moreover, industrially manufactured pharmaceuticals may contain different excipients, which may be either untested or not licensed for use in neonates. The aim of our study was to determine the prevalence and pattern of pharmaceutical and excipient exposure in newborns hospitalized at the Department of Neonatology, Ljubljana, Slovenia. METHODS: A longitudinal prospective cross-sectional study was performed during a one-month period and included all hospitalized neonates. Route of administration, site of action, type of manufacture, licensing status, type and concentrations of excipients for all pharmaceuticals given to the neonates were determined. RESULTS: Twenty seven different pharmaceutical preparations were prescribed to a total of 48 hospitalized newborns. In most cases, newborns were prescribed various pharmaceuticals that were not approved for use in this population. Newborns were exposed to 60 different excipients in industrially manufactured pharmaceutical preparations. More than half of the received pharmaceuticals contained potentially harmful and harmful excipients. CONCLUSIONS: Two-thirds of pharmaceutical preparations for neonates were used off-label. Newborns receive more auxiliary substances, which may be unsuitable for this age group and may even be toxic to them, via industrially manufactured pharmaceuticals.

Regional characteristics of histamine uptake into neonatal rat astrocytes.

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na(+)-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na(+). Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na(+)-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na(+)-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.

Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes.

Astrocytes have a key role in the clearance and inactivation of histamine in the adult central nervous system, but transporters which mediate histamine uptake into astrocytes have not been fully characterized. We therefore investigated the kinetic and molecular characteristics of histamine uptake into cultured adult rat astrocytes. [(3)H]-histamine was taken up by astrocytes in a temperature-, time- and concentration-dependent manner and was inhibited up to 60-70% by 1mM ouabain or by substitution of NaCl with choline chloride. Specific [(3)H]-histamine uptake, determined as the difference between transport at 37 and 4°C, displayed saturation kinetics with the apparent Michaelis-Menten constant (K(m)) of 141 and 101µM and the apparent maximal uptake rate (V(max)) of 22.5 and 17.8pmol/min/mg protein, as estimated from the Woolf and the Eadie-Hofstee plots, respectively. Since our data suggested the presence of a carrier-operated histamine uptake system, we assessed the possible involvement of the organic cation transporters (OCT) 1, 2 and 3, which have been previously described to play a role in histamine transport in the central nervous system. Low level mRNA expression of all OCT isoforms was detected, but in contrast to rat brain cortex homogenate, where OCT3 was the most prominently expressed OCT isoform, OCT2 mRNA was the predominant OCT species in cultured astrocytes. However, OCT inhibitors corticosterone and decynium 22 (D22) had no effect or only modestly reduced [(3)H]-histamine uptake. Thus, our data indicate that adult rat astrocytes possess an efficient high-capacity, low-affinity carrier-operated histamine uptake system, which does not seem to involve OCTs.

Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma.

Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.