Liposomal Carrier Research Articles

Light induced cytosolic drug delivery from liposomes with gold nanoparticles

Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells.

XXI век: как изменились наши представления о липосомальных лекарственных средствах

В обзоре представлена современная информация о разработке наноразмерных переносчиков лекарственных средств на примере липосом. Кратко изложены хронологические аспекты изучения липосом, включая их строение, состав, взаимодействие с молекулами и использование в качестве резервуарных систем доставки лекарств. Рассмотрены медицинские преимущества липосомальных препаратов по сравнению с применением традиционных лекарственных средств. Обсуждаются приёмы повышения эффективности включения лекарственного соединения в липосомы и стабилизации липосомальных лекарств, а также механизмы их взаимодействия с клетками.

Intravenous siRNA Silencing of Survivin Enhances Activity of Mitomycin C in Human Bladder RT4 Xenografts

Survivin inhibits apoptosis and enables tumor cells to escape from therapy induced senescence. High survivin expression is associated with bladder cancer aggressiveness and recurrence. We evaluated whether survivin expression is reduced by siRNA and whether survivin silencing would enhance mitomycin C activity in human RT4 bladder transitional cell tumors in vitro and in vivo. We assessed the effectiveness of siRNA therapy using 2 newly developed pegylated cationic liposome carriers, PCat and PPCat. Each has a fusogenic lipid to destabilize the endosomal membrane. PPCat further contains paclitaxel to enhance in vivo delivery and transfection of survivin siRNA. In vitro antitumor activity was evaluated by short-term MTT and long-term clonogenicity cytotoxicity assays. In vivo intravenous therapy was assessed in mice bearing subcutaneous tumors. Nontarget siRNA showed no antitumor activity in vitro or in vivo. Treatment of cultured cells with mitomycin C at a 50% cytotoxic concentration enhanced survivin mRNA and protein levels. Adding PPCat or PCat containing survivin siRNA reversed survivin induction and enhanced mitomycin C activity (p <0.05). In tumor bearing mice single agent mitomycin C delayed tumor growth and almost tripled the survivin protein level in residual tumors. Adding PPCat-survivin siRNA, which alone resulted in a minor survivin decrease of less than 10%, completely reversed mitomycin C induced survivin and enhanced mitomycin C activity (p <0.05). Results indicate that there is effective in vivo survivin silencing and synergism between mitomycin C and PPCat-survivin siRNA. This combination represents a potentially useful chemo-gene therapy for bladder cancer.

A study on the encapsulation of an occludin lipophilic derivative in liposomal carriers

Many peptides and proteins, although potentially useful for the treatment of various diseases, are hindered in their clinical use by poor oral absorption and rapid enzymatic degradation. One of the available solutions to these problems is to increase the lipophilicity by conjugating the peptides to lipophilic moieties, making them more able to cross the biomembranes by passive transport. Occludin is a 65-kDa integral plasma-membrane protein located at the tight junctions. This protein and the peptide derived from it have potential clinical application for drug delivery. Peptide OP90-103 (1) is a fragment of occludin that shows a very poor oral bioavailability and is highly susceptible to enzymatic degradation. The conjugation of 1 with two lipoamino acid (LAA) moieties has been shown to enhance its lipophilicity and bioavailability, as well as its enzymatic stability. The purpose of this study was to evaluate the possibility of encapsulating fluorescein modified lipidated OP90-103 (2), in unilamellar- (LUV) and multilamellar liposomes (MLV), which have a different composition and surface charge and are produced by different methods. The cell internalization of the carrier systems was evaluated in vitro.

Surface-modified Epirubicin-HCl liposomes and its in vitro assessment in breast cancer cell-line: MCF-7

Background: Epirubicin-HCl is highly efficient for breast cancer management at a concentration of 60–90 mg/m2. However, its application is limited due to cumulative dose-dependent cardio-toxicity.Purpose: The main aim of this study was to formulate breast cancer-targeted liposomal carrier by surface conjugation of transferrin to minimize cardio-toxicity of drug along with improved pharmacokinetic profile.Method: Liposomes were formulated by ethanol injection method using HSPC, cholesterol and DSPG and later loaded with drug by the ammonium sulfate gradient method. The formulation was characterized for physicochemical properties like size, zeta potential, entrapment efficiency, TEM; in vitro tests like electro-flocculation, hemolysis and drug release; cell line study (MCF-7 cells); in vivo studies including LD50 determination, pharmacokinetic analysis, myocardial toxicity determination and stability.Results and discussion: Optimized formulation had molar ratio of 60:30:8:2 (HSPC:Chol:DSPG:mPEG-DSPE) with entrapment efficiency ∼83%, particle size below 200 nm and zeta potential about −20 mV. In vitro studies proved non-interfering property and drug release character of formulation while cell line studies demonstrated improvement in cell uptake and thereby increased cytotoxicity of targeted formulation. The IC50 value obtained for epirubicin solution, non-targeted and targeted liposomes was 0.675, 0.532 and 0.192 µg/ml, respectively. Furthermore, in vivo tests validated safety and distribution profile of prepared formulations.Conclusion: Apt properties of prepared Epirubicin-HCl liposomal formulation warrant its clinical application in breast cancer treatment after further studies.

Lipid/detergent mixed micelles as a tool for transferring antioxidant power from hydrophobic natural extracts into bio-deliverable liposome carriers: the case of lycopene rich oleoresins

Lipid/detergent mixed micelles promote and modulate the incorporation of carotenoids from natural oleoresins into bio-deliverable liposome carriers.

Modulation of induced cytotoxicity of doxorubicin by using apoferritin and liposomal cages.

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin (“Apodox” and “lip-8-dox”) and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL‑2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

Formulation and In Vitro Characterization of Thermosensitive Liposomes for the Delivery of Irinotecan

The effect of the lipid composition on the physicochemical properties of a liposomal carrier containing irinotecan (CPT-11) and its in vitro antitumoral activity on a colon cancer cell line has been investigated. The paper describes the procurement of a novel and easy-to-prepare temperature-sensitive carrier for CPT-11 and proves its suitability as delivery system on the basis of its ability to incorporate the drug and on its efficiency to promote drug internalization. Permeability data, studied in vitro in a simulated biological medium at different temperatures, showed that both the nature of the lipids and the ratio in which they have been mixed play a key role in the release of the encapsulated product. Stable temperature-sensitive liposomes with good drug incorporation efficiency were obtained and characterized. The cellular uptake and the cytotoxic activity of the CPT-11 liposomal formulation were compared with those corresponding to the free drug with promising results, being concentration and time dependent and significantly higher. Thus, it could be interpreted that the greatest cytotoxic effect of liposomal CPT-11 was because of the increased uptake provided by the liposomal carrier. Studies of cell cycle and annexin V binding showed drug-induced changes in cell cycle dynamics and the proapoptotic effect of liposomal CPT-11.

Quantitative evaluation of DNA dissociation from liposome carriers and DNA escape from endosomes during lipid-mediated gene delivery.

Nonviral vectors are highly attractive for gene therapy from a clinical point of view, and cationic lipid nanoparticles in particular have generated considerable interest. However, despite considerable recent advances, problems associated with low transfection efficiencies remain to be resolved to fully meet the potential of these vectors. The trafficking of plasmid DNA (pDNA) from the extracellular space up to the nucleus is prevented by several barriers, including liposome/pDNA dissociation within the endosome and pDNA escape into the cytosol. The aim of this work was to develop and optimize a tool that could offer simultaneous quantitative information both on the intracellular dissociation of oligonucleotides from lipid nanoparticles, and on the DNA escape from endocytic compartments. The ability to follow in real time both of these processes simultaneously (in a quantitative manner) is expected to be of high value in the rationalization and conception of new lipid nanoparticle vectors for gene delivery for therapeutic purposes. To this effect, a combination of Förster resonance energy transfer (FRET) and colocalization microscopy was employed. We show that it is possible to distinguish between liposome/pDNA dissociation and depletion of DNA within endosomes, providing resolution for the detection of intermediate species between endocytic particles with intact lipoplexes and endosomes devoid of DNA because of DNA escape or degradation. We demonstrate that after endocytosis, exceptionally few endocytic particles are found to exhibit simultaneously DNA/lipid colocalization and low FRET (DNA/lipid dissociation). These results clearly point to an extremely short-lived state for free plasmid within endosomes, which either escapes at once to the cytosol or is degraded within the endocytic compartment (because of exposure of DNA). It is possible that this limitation greatly contributes to reduction in probability of successful gene delivery through cationic lipid particles.

Designing a better theranostic nanocarrier for cancer applications.

Nanocarriers show incredible potential in theranostic applications as they offer diagnostic capabilities along with the ability to encapsulate and protect drugs from degradation, be functionalized with targeting moieties and be designed with controlled release mechanisms. Most clinically approved nanocarrier drugs are liposomal formulations. As such, considerable research has been directed towards designing liposomal carriers that can release their payloads via exogenous or endogenous triggers. For triggered release to effectively increase drug bioavailability, nanocarriers must first accumulate at the tumor site via the enhanced retention and permeability effect. It has been demonstrated in the chicken embryo chorioallantoic membrane and murine xenografted models that nanoparticle surface charge and geometry, with respect to vascular endothelium fenestration size, drive this accumulation in angiogenic tissue.

Cationic liposome-nucleic acid complexes for gene delivery and gene silencing.

Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL-nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL-nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL-DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure-function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL-DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications.

Cationic surface charge enhances early regional deposition of liposomes after intracarotid injection.

Rapid first pass uptake of drugs is necessary to increase tissue deposition after intraarterial (IA) injection. Here we tested whether brain tissue deposition of a nanoparticulate liposomal carrier could be enhanced by coordinated manipulation of liposome surface charge and physiological parameters, such as IA injection during transient cerebral hypoperfusion (TCH). Different degrees of blood-brain barrier disruption were induced by focused ultrasound in three sets of Sprague-Dawley rats. Brain tissue retention was then compared for anionic, cationic, and charge-neutral liposomes after IA injection combined with TCH. The liposomes contained a non-exchangeable carbocyanine membrane optical label that could be quantified using diffuse reflectance spectroscopy (DRS) or visualized by multispectral imaging. Real-time concentration-time curves in brain were obtained after each liposomal injection. Having observed greater tissue retention of cationic liposomes compared to other liposomes in all three groups, we tested uptake of cationic liposomes in C6 tumor bearing rats. DRS and multispectral imaging of postmortem sections revealed increased liposomal uptake by the C6 brain tumor as compared to non-tumor contralateral hemisphere. We conclude that regional deposition of liposomes can be enhanced without BBB disruption using IA injection of cationic liposomal formulations in healthy and C6 tumor bearing rats.

Matrix liposomes: A solid liposomal formulation for oral administration

Liposomes can be used as oral dosage form to improve the bioavailability of both hydrophilic drugs, such as peptides and proteins and lipophilic drugs. But liposome dispersions are not very stable under the harsh conditions of the GI‐tract. Also a controlled release of embedded compounds is not possible. Therefore we developed a lipid based liposomal carrier system for peroral delivery of peptides. The lipid bilayer of standard egg‐PC/cholesterol liposomes were stabilized by gelatine. The gelatine also works as a thickening agent by forming a matrix in which liposomes are embedded. Liposomes were prepared by dual asymmetric centrifugation with the addition of gelatine. Size and size distribution of prepared liposomes with up to 20% gelatine achieved the same quality compared to conventional liposomes. Size and size distribution, encapsulation efficiency of different compounds and the dissolution behaviour of the solid dosage form in simulated intestinal fluid were examined. Matrix liposomes containing 20% gelatine showed an approximately linear release of liposomes. Moreover, the dissolution rate could be adjusted by modification of the gelatine concentration and the absolute amount of liposomes released at a certain time was found to be dose dependent.Practical applications: The new liposomal solid formulation can be useful for a controlled release of hydrophilic macromolecules as peptides and proteins after peroral administration. Liposomes embedded in a solid dosage form offer the possibility of a simple administration and exhibit higher storage stability compared to liposomes in dispersion.A composition of phospholidpids and gelatine leads to matrix liposomes prepared by dual asymmetric centrifugation. Dissolution of the solid liposomal formulation leads to a liposome release out of the dosage form. The dissolution rate can be adjusted by the concentration of gelatine.

Nanodrug-enhanced radiofrequency tumor ablation: effect of micellar or liposomal carrier on drug delivery and treatment efficacy.

PurposeTo determine the effect of different drug-loaded nanocarriers (micelles and liposomes) on delivery and treatment efficacy for radiofrequency ablation (RFA) combined with nanodrugs.Materials/MethodsFischer 344 rats were used (n = 196). First, single subcutaneous R3230 tumors or normal liver underwent RFA followed by immediate administration of IV fluorescent beads (20, 100, and 500 nm), with fluorescent intensity measured at 4–24 hr. Next, to study carrier type on drug efficiency, RFA was combined with micellar (20 nm) or liposomal (100 nm) preparations of doxorubicin (Dox; targeting HIF-1α) or quercetin (Qu; targeting HSP70). Animals received RFA alone, RFA with Lipo-Dox or Mic-Dox (1 mg IV, 15 min post-RFA), and RFA with Lipo-Qu or Mic-Qu given 24 hr pre- or 15 min post-RFA (0.3 mg IV). Tumor coagulation and HIF-1α orHSP70 expression were assessed 24 hr post-RFA. Third, the effect of RFA combined with IV Lipo-Dox, Mic-Dox, Lipo-Qu, or Mic-Qu (15 min post-RFA) compared to RFA alone on tumor growth and animal endpoint survival was evaluated. Finally, drug uptake was compared between RFA/Lipo-Dox and RFA/Mic-Dox at 4–72 hr.ResultsSmaller 20 nm beads had greater deposition and deeper tissue penetration in both tumor (100 nm/500 nm) and liver (100 nm) (p<0.05). Mic-Dox and Mic-Qu suppressed periablational HIF-1α or HSP70 rim thickness more than liposomal preparations (p<0.05). RFA/Mic-Dox had greater early (4 hr) intratumoral doxorubicin, but RFA/Lipo-Dox had progressively higher intratumoral doxorubicin at 24–72 hr post-RFA (p<0.04). No difference in tumor growth and survival was seen between RFA/Lipo-Qu and RFA/Mic-Qu. Yet, RFA/Lipo-Dox led to greater animal endpoint survival compared to RFA/Mic-Dox (p<0.03).ConclusionWith RF ablation, smaller particle micelles have superior penetration and more effective local molecular modulation. However, larger long-circulating liposomal carriers can result in greater intratumoral drug accumulation over time and reduced tumor growth. Accordingly, different carriers provide specific advantages, which should be considered when formulating optimal combination therapies.

Investigation of Drug-Excipients Compatibility of Ellagic Acid for Development of Formulation Containing Lipososmes

The aim of the present study was tochecked the compatibility between drug: lipid and drug: excipients. Ellagic acid was selected as model drug while phospholipid and Cholesterol were considered excipients. The study was performed by Fourier transform infrared spectroscopy (FTIR), Diffraction scanning calorimetric (DSC) and High performance liquid chromatography (HPLC). The spectrophotometric graphs revealed that there was no significant changes in the position of functional groups of ellagic acid, phospholipid and Cholesterol (O-H, C=O, C-H str.) in pure drugs and excipients with respect to their physical mixtures. Further the samples werecharacterized by using DSC and HPLC methods and found no any interaction between drug excipients. Combination of synthetic herbal drug and pharmaceutical excipients are compatible with each other and shows no interaction. Thus this suitable combination can be employed for the successful development of novel liposomal carrier system.

Effect of 5-aminolevulinic acid-encapsulate liposomes on photodynamic therapy in human cholangiocarcinoma cells.

Intracellular protoporphyrin IX (PpIX) generation following administration of 5-aminolevulinic acid (ALA) has been used in photodynamic therapy (PDT). Subsequent irradiation can lead to selective damage to photosensitizer-treated cells or tissues. In the present work, we describe the enhancement of ALA-induced PpIX accumulation using a liposome carrier. ALA-containing liposomes (Lipo-ALA) were prepared using dipalmitoyl-phosphatidyl choline and in vitro PDT effect was investigated against human cholangiocarcinoma HuCC-T1 cells. Lipo-ALA increased the uptake efficiency into tumor cells compared to ALA itself, which increased the phototoxic effect. A positive relationship was evident between small particle size, PpIX accumulation and cell death after Lipo-ALA based PDT.

Preclinical studies of pegylated- and non-pegylated liposomal forms of doxorubicin as radiosensitizer on orthotopic high-grade glioma xenografts.

Free doxorubicin (DXR) is not currently used to treat brain tumors because (i) the blood-brain barrier limits the drug deposition into the brain (ii) lethal toxic effects occur when combined with radiation therapy. Since encapsulation of DXR within liposomal carriers could overcome these drawbacks, the present study aimed at evaluating the radiosensitizing properties of non-pegylated (NPL-DXR) and pegylated (PL-DXR) liposomal doxorubicin on orthotopic high-grade glioma xenografts (U87). DXR accumulation in brain tissues was assessed by a high-performance liquid chromatography method and antitumor efficacy was evaluated by mice survival determination. We showed that encapsulation of DXR ensured a preferential deposition of DXR in tumoral tissue in comparison with normal brain tissue: the best AUC tumor tissue/AUC normal tissue ratio depended greatly on the schedule. Overall, thanks to the optimization of the delivery schedule, we demonstrated a radiosensitizing effect for both liposomal DXR without toxicity of this combination on the U87 human malignant glioma orthotopic xenografts. This study shows that the use of nanocarriers, allowing targeting of intracerebral tumor, renders relevant the combination of anthracyclin with radiation therapy to treat brain tumors, opening a new field of therapeutic applications. However, our results point out that, for each new delivery system, the administration schedules need to be rigorously optimized.

Development of a gradient high performance liquid chromatography assay for simultaneous analysis of hydrophilic gemcitabine and lipophilic curcumin using a central composite design and its application in liposome development

A rapid and reliable gradient high performance liquid chromatography (HPLC) method was developed for the simultaneous analysis of a hydrophilic anticancer drug, gemcitabine, and a lipophilic phytochemical, curcumin. A central composite design (CCD) was employed to optimize the gradient elution programme to obtain an acceptable retention time and peak width for curcumin with the aid of the desirability function (DF). Dual wavelengths (gemcitabine, 270nm; curcumin, 420nm) were selected to give maximal specific detection using a diode-array detector. The optimized DF was obtained with a mobile phase (water:acetonitrile) of 95:5 for 4.5min, followed by 30:70 for 5min, which gave retention times within 11min with peak widths <0.10min, respectively. No interference from the formulation ingredients was detected. The method was linear over the range of 0.125–5μM for gemcitabine, and 1.25–50μM for curcumin. The intra-day and inter-day variability were less than 5%, with accuracies between 90 and 110% of the true values. The limit of detection (LOD) for gemcitabine and curcumin was 0.04μM and 0.03μM, respectively. Using this method, release study for a dual-drug loaded formulation revealed a Fickian diffusion process for both. The stability of curcumin was pH dependent in aqueous solutions with a half-life of 3h at 25°C and pH 7.4. This was significantly improved after incorporation into the liposomes with no degradation for at least 3 months. In conclusion, the combination of CCD and DF offers a new approach to optimize a gradient HPLC method with high efficiency. A liposomal carrier system may provide an option for tumour dual-drug therapy with the additional advantage of chemical stabilization of the encapsulated compound.

Abstract 130: Immediate Stabilization of Subarachnoid Hemorrhage by Intranasal Delivery of Novel Therapeutics

Background: Stroke treatment is a challenge because of the short time window. Intranasal therapeutics delivery is a promising administration options for early delivery of brain stabilization agent as both brain and nasal compartments are interconnected via the olfactory/trigeminal structure and the peripheral circulation. Due to the stabilization effect of nitric oxide (NO) on endothelium, vascular smooth muscle, polymorphonuclear neutrophils (PMNs) in acute cellular and tissue injury, NO releasing agents has been used for emergent cardiovascular conditions such as acute cardiac ischemia and stroke. Here we investigated an intranasal carrier/NO therapeutic delivery strategy to the brain for acute treatment in hemorrhage stroke. Methods: NO/argon (1:9) in caged molecular, cyclodextrin, in liposomal formulation was created to carry NO. A total 200 ul (50 ul per 2 minutes х 4 times) of the NO liposomal formulation or a liposomal carrier control was administrated intranasally at 30 minutes after subarachnoid hemorrhage (SAH). Distribution of NO in brain was imaged with DAF-FM. Hemorrhagic volume was quantified by spectrophotometric hemoglobin assay two hours after SAH. Results: NO distribution was noted in normal brain tissue (Fig.1A). SAH rats had reduced NO distribution due to hemoglobin scavenging of NO (Fig.1B). Intranasal NO liposomal delivery enhanced NO distribution especially in the cerebral microvascular in SAH brain (Fig. 1C). Effective intranasal NO liposomal delivery resulted in decreased hemorrhagic volume (115 ± 22μl) vs SAH without treatment control (344 ± 63μl, p=0.014) as well as SAH with intranasal liposomes control (298 ± 114μl, p=0.02) (Fig. 1D). Conclusions: The Intranasal delivery route of liposomal carriers efficiently delivers therapeutics into the brain. These carriers have controlled release properties in addition to their therapeutic payload effects. The present method has great potential for immediate delivery in stroke.