As a self-assembled mimetic structure of biological membranes, polydiacetylene liposomes have been studied for the development of platforms for various applications including nano-containers, nano-transporters, and nano-delivery systems for biological-, life- and materials-science applications. Liposomes incorporating amphiphilic polymer poly(10, 12 pentacosadiynoic acid) (PDA) was used as a building block for investigations mimicking cellular reaction and processes at the membrane cell. Changes in local membrane micro-organization and packing as a result of biomolecular and bioparticle reactions and processes at the liposomal membrane were investigated through the use of colorimetric and emission responses of PDA liposomes in solution phase. My dissertation comprises of six chapters. I provide brief overview of each chapter in the following paragraphs: Chapter 1: Introduction. In this chapter, an introduction is given on structure and function of lipid bilayer and multilayer of liposomes from a perspective of shared features with biological membranes. Amphiphilic molecules along with natural lipids at (or higher) critical micelle concentration self-assemble in aqueous medium, thereby, forming a lipid bilayer or multilayer to reduce the free energy of the system. When one of the components of the lipid bilayer is a polymerizable monomer, micelles/liposomes with enhanced mechanical and chemical stability are achieved. The lipid bilayer of liposomes is a boundary that includes at least three different regions: inside aqueous cavity, hydrophobic membrane zone, and membrane-aqueous interfaces. The membrane surface is available for further functionalization. In general, all three regions of the liposomes are utilized for both fundamental and applied studies. For example, the PDA liposomes have been employed for biosensing, drug/protein/nucleic acid transport and delivery and target release, and various probing cellular-like reactions and processes at the membranes. Here, in this chapter, literature on PDA was reviewed for a time period of 2008-2015. Furthermore, emphasis was given to application of PDA liposomes as (bio) sensing elements utilizing colorimetric, fluorescence, and FRET mechanisms. Chapter 2. Polydiacetylene (PDA) liposomes have been accepted as attractive colorimetric bionanosensors. The molecular recognition elements, either embedded within the liposomal membrane or covalent bound at the membrane surface, are available for interaction with biological and chemical analytes. Usually, PDA liposomes perform transduction activity through perturbation of the conjugated polymer backbone, which provides a colorimetric change in solution or solid-state phase. Here, we report that trapping self-quenched fluorescent specie within inner cavity of the liposomes is a simple and effective analytical tool for evaluating biomolecular binding events at the membrane surface. The release of fluorophores in response to the membrane binding event led to amplified emission signal which was utilized for probing reactions at the membrane surface that mimics reactions occurring at the cellular membrane surface. Specifically, a covalent binding on enzyme-substrate reaction resulted in a change of membrane fluidity, thereby releasing inner fluorophore content of the PDA liposomes. Fluorescent markers were loaded at or higher self-quenched concentration in the cavity of the liposome. Amplification of the fluorescence intensity was positively correlated with the concentration of protein added in the solution. The bilayer fluidity alteration also appears to depend on the molecular weight of the protein bound at the membrane. Overall, binding of protein with membrane promoted changes in the local PDA membrane organization and packing that enhanced the membrane permeability. The encapsulated content therefore leaked through “transient pores” formed in the membrane yielding substantial emission amplification. Chapter 3. Inspired by stability of the PDA liposomes, surface functionalization with a variety of molecules and loading within bilayer and inner cavity of the liposomes, we utilized liposomes as biocatalytical nanoreactors. Removable template molecules were embedded in the lipid bilayer and active protein encapsulated in the internal cavity was used for studying the transport properties of liposomes through substrate-enzyme reactions. Detergent Triton X-100 was used to remove a small portion of lipid and template molecules embedded in the membrane. The removal of lipid/template molecules not only affected the membrane fluidity but also provided transient pores in the membrane, allowing transport of substrate for enzymatic oxidation of glucose and 2-deoxy-glucose. Three important biological-relevant properties of cellular membrane: transport, bioavailability, and bio-reactivity of enzyme and substrate were studied. We found that enzyme molecules retained their reactivity when encapsulated within the aqueous inner cavity of the PDA liposomes, and that their activity was comparable to that in the bulk solution. Chapter 4. This chapter introduces studies on (at least partially) answering important questions how and if anchored enzyme activity at the liposome surface is affected through limited diffusion and spatial constraints. A further crucial question was investigated what effect of protein binding at the surface of the liposomes to enzymatic activity was. These relevant questions were important for increasing our fundamental knowledge related to reactions, interactions, and transport processes in biological cellular systems. A functionalized liposome system containing enzyme (Trypsin) covalently attached at the PDA liposome surface was synthesized. Using PDA liposomes as an immobilization scaffold, we evaluated and compared the cleavage behaviors of Trypsin in either immobilized at the membrane surface or in the free form. The covalent binding interaction and tryptic cleavage at the membrane-water interface was monitored by UV-vis and fluorescent spectroscopy, fluorescent anisotropy and spectro-micro-imaging. Trypsin binding at the membrane appeared to be significantly affected the enzymatic activity of the bound enzyme as seen from colorimetric response of the PDA liposomes. Chapter 5. Hierarchical structures support structures with new functionalities, therefore, advances in fabrication and characterization of biomimetic systems based on biological building blocks may present substantial potential rewards in material science. We take advantage of non-covalent forces known in biology for creating spatial organization by assembly tobacco mosaic virus-liposome polymeric hierarchical systems through biotin-streptavidin linkages. The advantage of using the biological thin rods such as TMV is that it can span the whole liposomal membrane allowing us to create microscopic hinge structures that connected liposomes. Our findings through electron and fluorescence microscopy confirmed that SA-TMV motif was able to stay inserted within the lipid bilayer of liposomes which yielded hierarchical structures after binding with Bt-liposomes. These hierarchical structures may find potential applications in targeted load (drug/protein/DNA) delivery, investigations involving virus-cell interactions, and sensing of virus particles. Chapter 6. Conclusions and Future work The present work in this dissertation utilized exploitation of biological self-assembly of small lipid molecules and larger biological-like motifs for enhancing our understanding of reactions and processes occurring at the cellular membrane surface. Overall the following four major studies were accomplished; 1. Sensing through amplified delivery, 2. Triggering an encapsulated bioreactor system at nanometric size, 3. Holding active biological elements when liposomes perform an attachment matrix, 4. Formation of hierarchical structures promoted by self-assembling of biological motifs with mimickers of cell membrane From our findings by mimicking the lipid bilayer of cell structures through liposomal membrane future work holds different ways to contribute in enhancing fundamental understanding of biological behavior. Active transport is an important function of all natural cells, playing important roles in intercellular communication. Liposomes composed of natural and polymerizable lipids may allow investigation involving exocytosis, formation of filopodia, vesicle fusion, budding and reproduction of neural synapses. Our liposome system may also mediate a broader range of highly selective and sensitive detection and sensing of cellular reactions and processes in physiological condition. I hope that this work in collaboration with multiple PIs will contribute to the fields at the interface of biology and material science.

Dielectrophoresis and electrorotation are receiving increasing attention as useful phenomena for the characterisation and physical manipulation of cells. The primarily concern of this investigation was to determine whether these techniques can interpret accurately the dielectric properties of biological cells with an appropriate dielectric shell model. In this study, synthetic vesicles have been used as the testing samples in electrorotation experiments to verify the reliability of these techniques. By using the electrorotation technique with the dielectric shell models, dielectric properties of vesicles could be analysed very accurately and the results were in agreements with the observed morphology and membrane properties. The physical structure of the vesicles varied from a simple one such as unilamellar vesicle, to a more complex structure such as the oligolamellar and multilamellar form. The morphology and membrane structure of the vesicles were also characterised by fluorescence microscopy, flow cytometry and electron spin resonance using spin probes. This allowed this validification of the application of the dielectric shell theory for analysis of simple cellular systems. The second objective of this work was to extend the potential used of the electrorotation technique, and not only for the analysis of cellular systems. Electrorotation was also performed on single stranded DNA oligonucleotides, covalently bound onto the surface of microscopic-sized latex beads. Different types of the DNA oligonucleotides exhibited different electrorotation responses according to their different base sequences. This has shown that the electrorotation technique can be used as an analytic tool to identify different sequences of DNA oligonucleotide.

Orientador: Maria Cristina Volpato, Francisco Carlos Groppo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T03:44:20Z (GMT). No. of bitstreams: 1 Caldas_CristinaSaragiotto_M.pdf: 985125 bytes, checksum: b1fed0990a0c2cfad07e069fd573fcc8 (MD5) Previous issue date: 2012 / Resumo: Este estudo teve como objetivo avaliar a eficácia anestésica de duas formulações de mepivacaína encapsulada em lipossomas (uni e multilamelar) com concentração lipídica 8 mM, comparando-as com solução de mepivacaína com epinefrina em três modelos: bloqueio do nervo infraorbital (BNIO), bloqueio do nervo alveolar inferior (BNAI) e infiltração subcutânea em ferida cirúrgica (ISFC), em ratos. Em cada experimento os animais receberam injeção de uma das seguintes formulações: mepivacaína 2% associada à epinefrina 1:100.000 (Mepi-Epi), mepivacaína 3% lipossomal unilamelar (Mepi-LUV) e mepivacaína 3% lipossomal multilamelar (Mepi-MLV) e os respectivos controles: solução de NaCl 0,9%, suspensão lipossomal unilamelar e suspensão lipossomal multilamelar sem anestésico. Para o BNIO (30 ratos) e BNAI (51 ratos) os animais foram divididos em 3 grupos, e receberam uma das formulações contendo mepivacaína no lado direito e a respectiva formulação controle no lado esquerdo, sendo respectivamente 0,1 mL no BNIO e 0,2 mL no BNAI. A s o N oram avaliados dura ão e sucesso da anestesia no l bio su erior, por pinçamento. Após o NA oram avaliados latência, dura ão e sucesso da anestesia ul ar or estímulo el trico. Para a ISFC, 48 ratos foram divididos em 6 grupos, sendo submetidos a estímulo inflamatório na pata traseira direita (incisão e sutura). Após 24h os animais que apresentaram hipernocicepção (diminuição de pelo menos 20% na sensibilidade à pressão - analgesímetro de von Frey) receberam injeção de 0,1 mL de uma das formulações, contendo mepivacaína ou controle, ao lado da ferida cirúrgica. A anestesia local foi avaliada com analgesímetro de von Frey a cada 10 minutos. Os resultados foram submetidos aos testes Log-Rank, Tukey, Kruskal-Wallis, Student-Newman-Keuls (alfa=5%). Nos três modelos estudados, a solu ão de Me i-Epi promoveu maior sucesso e duração da anestesia (p<0,05), enquanto as formulações lipossomais não diferiram entre si (p>0,05). Para o experimento do BNAI, não foram observadas diferenças entre as formulações (p>0,05) para latência da anestesia. Conclui-se que a encapsulação em lipossomas uni ou multilamelares foi menos eficaz do que a epinefrina em aumentar a eficácia anestésica da mepivacaína em todos os modelos estudados / Abstract: The aim of this study was to evaluate the anesthetic efficacy of two liposomal (unilamelar and multilamelar, 8mM lipid concentration) mepivacaine formulations compared to mepivacaine with epinephrine in three models: infraorbital nerve block (IONB), inferior alveolar nerve block (IANB) and subcutaneous infiltration in surgical wound (SISW) in rats. In each model animals received one of the following formulations: 2% mepivacaine with 1:100,000 epinephrine, (Mepi-Epi), liposomal unilamelar 3% mepivacaína (Mepi-LUV), liposomal multilamelar 3% mepivacaína (Mepi-MLV), and the respective controls: 0.9% NaCl solution, and liposomal unilamelar and liposomal multilamelar suspensions without the local anesthetic. For IONB (n= 30) and IANB (n= 51) the animals were divided into 3 groups, which received one of the formulations containing mepivacaine in the right side and the respective control formulation in the left side; 0.1 mL and 0.2mL were respectively injected for IONB and IANB. After IONB the success and duration of anesthesia was evaluated by upper lip pinching.After IANB the onset, duration and success of pulpal anesthesia were evaluated by electric stimulus. For SISW experiment the 48 rats were divided into 6 groups and submitted to an inflammatory stimulus in the right hind paw (incision and suture). After 24 h the animals that presented hypernociception (at least 20% decrease in the baseline response to force - von Frey anesthesiometer) received a 0.1mL injection of one of mepivacaíne or control formulations at the side of the surgical wound. Local anesthesia was evaluated each 10 min with von Frey anesthesiometer. Data were submitted to Log-Rank, Tukey, Kruskal-Wallis and Student-Newman-Keuls tests (alpha=5%). Mepi-Epi provided higher success and duration of anesthesia (p<0.05) in the three studied models; no difference was observed between the liposomal formulations (p>0.05). No differences in the anesthesia onset were observed among formulations for IANB (p>0.05). It is concluded that the encapsulation in unilamelar and multilamelar liposomes was less effective than epinephrine to improve mepivacaine anesthetic efficacy in all studied models / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

Acyclovir is a synthetic purine nucleoside analogue with in vitro and in vivo inhibitory activity against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella-zoster virus (VZV). The efficacy of oral acyclovir is limited as a result of its low bioavailability (15-30%) as it is poorly water soluble and therefore requires a frequent dosing regimen. When orally administered, peak plasma concentration occurs after 1.5–2.5 hours, while its elimination half-life is approximately 2-3 hours. Acyclovir displays poor solubility in water and in lipid bilayers, which leads to poor drug levels at target sites after oral, local, or parenteral administration. In order to improve this lack of solubility, novel amphiphilic derivatives have been designed to form nanoparticles, which allow for the efficient encapsulation of this hydrophobic antiviral agent. Reformulation of drugs in liposomes has provided an opportunity to enhance the therapeutic indices of various agents mainly via alteration of their bio-distribution. Liposomal drug delivery systems have received considerable attention due to their immense advantages which include, effective encapsulation of both small and large molecules that have a wide range of hydrophobicity levels and pKa values, prolonging and targeting release of therapeutic agents by modification of liposomal surface and also minimising clinical drug dose thus reducing toxicity effects. Liposomes exhibit a number of special biological characteristics, including specific interactions with biological membranes and various cells, hence, liposomes are used as biocompatible carriers to improve delivery properties across mucus membranes. Mucoadhesive dosage forms may be designed to enable prolonged retention at the site of application, providing a controlled rate of drug release for improved therapeutic outcome. The aim of this study was to develop an acyclovir liposomal mucoadhesive film by actively encapsulating acyclovir into liposomes and preparing a mucoadhesive film to optimise delivery of acyclovir liposomes at target sites. To actively encapsulate acyclovir and prepare the acyclovir-containing liposomes, a comprehensive statistical methodology was used in optimising the liposome formulation to encapsulate acyclovir. Central composite design was used as the response surface methodology statistical tool to design and develop an optimised method for active encapsulation of acyclovir into liposomes. The predicted optimised encapsulation parameters were incubation temperature of 60 °C and incubation time of 45 minutes. The mean percentage encapsulation calculated was 27.72%. The overall average size of the liposomes was 99.5 nm with a narrow distribution polydispersity index of 0.105 and were physically characterised as small unilamellar vesicles which possessed an average zeta potential of -45.6 mV. High Performance Liquid Chromatography (HPLC) was used to analyse and determine acyclovir drug content in the liposomes and drug release pattern from the mucoadhesive film. Polyvinyl-pyrrolidone (PVP) and Polyethylene glycol (PEG) were used in the preparation of mucoadhesive film, in which the acyclovir encapsulated liposomes were incorporated. The average amount of acyclovir drug content quantified to be in 4 cm2 of the mucoadhesive film was 36.8543 μg. The average tensile strength of the mucoadhesive film was determined to be 3.06 N/mm2 with an elongation percentage of 4.54%. The toughness of the film was 71.50 N.mm and the force required to rupture film was 16.49 N. The work and maximum force required to detach the mucoadhesive film from the glass side was 2.58 N.mm and 11615.32 mN, respectively. A Franz diffusion cell was used to perform acyclovir drug release studies from the mucoadhesive film and a commercial brand of acyclovir cream (Acitop®). Percentage acyclovir drug release from the film and cream was plotted against time using Sigmaplot® software version 13 following First order, Peppas, Hixon and Crowell, Higuchi (Square Root Time) and Bakers and Lonsdale mathematical models. The mucoadhesive film acyclovir attained the highest correlation coefficient r2 of 0.9879 following the Baker & Lonsdale mathematical model which describes controlled drug release from spherical matrices hence fits the model as the acyclovir is encapsulated in liposomes which are incorporated in the polymer mucoadhesive film. And the acyclovir cream (Acitop®) attained the highest correlation coefficient r2 of 0.9944 following the Peppas mathematical model. The Peppas model has been used to describe drug release from various release dosage forms when there is more than one type of dosage release or when release mechanism is not well known. One assumption of this model is that drug release occurs in one dimension, which is a suitable release profile for the cream as it is absorbed through the skin in one dimension when applied topically. There was significant difference between the drug release data for the mucoadhesive film and the acyclovir cream (Acitop®). A physically stable mucoadhesive film containing acyclovir-loaded liposomes was developed.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Veronika Skalická Supervisor: Dr. Georgios Paraskevopoulos, Ph.D.; Michael Binnefeld Title of Diploma thesis: Up-scaling and further development of matrix liposomes The thesis evaluates novel matrix liposomes formed by dual asymmetric centrifugation technology. Particles' parameters (size, size distribution and ζ-potential) were analyzed by dynamic light scattering method using ZetaSizer device. Encapsulation efficacy was determined by size exclusion chromatography using carboxyfluorescein as fluorescent encapsulation marker. The overall results support that the most beneficial formulation parameters are 15 min of speed mixing process with ceramic beads of diameter 1,0-1,2 mm. Up-scaling of the procedure didn't drastically affect liposomal parameters up to a lipid batch load of 700 mg. Furthermore, the usage of different types of gelatin or glycerinated gelatin didn't significantly influenced particles' characteristics. It is noteworthy that the matrix composed of 50% glycerinated gel sustained the ability to form liposomes by dispersion of vesicular phospholipid gels in phosphate buffer saline even after 31 days. Other matrices showed a damage of liposomes as confirmed by size and size...

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Immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through the triggering of the endosomally localized TLR9 by a poorly understood mechanism. The immunopotency and broad range of activity of CpG ODN makes it a promising immunotherapeutic for the treatment and prevention of cancer and other diseases. However, rapid degradation of ODN by serum nucleases, low levels of accumulation in target tissue and lack of specificity for and poor uptake into target cells following systemic administration pose significant hurdles for the clinical application of CpG ODN. This thesis describes the immunostimulatory properties of CpG ODN encapsulated in liposomal nanoparticles (LN), a delivery system that overcomes many of the problems impeding the clinical development of "free" ODN. In particular, it is shown that LN delivery of CpG ODN specifically targets the ODN for uptake by immune cells in vivo, providing a basis for significantly enhanced immunostimulatory activity, including more potent innate and adaptive immune responses, that ultimately improve anti-tumour efficacy. A particular focus of this thesis concerns previous observations that methylated sequences in ODN (mCpG ODN) are immunologically inert. It is shown that encapsulation of mCpG ODN in LN results in immunostimulatory activity that is equal to or greater than that observed for LN formulations of the equivalent unmethylated form as judged by various immune parameters and anti-tumour efficacy. Further, it is shown that both LN-mCpG ODN and LN-CpG ODN exert their immunostimulatory effects via TLR9 based on preliminary in vitro results and confirmed by studies performed in TLR9 knockout animals. The mechanisms responsible for the differentiation between both CpG ODN and mCpGODN and how encapsulation endows immunostimulatory potential are explored. It is shown that discrimination occurs upstream of TLR9 and that the lack of immunological activity of free mCpG ODN is not due to differences in uptake, trafficking to endosomal compartments or ability to bind to TLR9, when compared with CpG ODN, but rather due to its ability to colocalize with TLR9 in the endosomal compartment. It is proposed that whereas the uptake of free CpG ODN results in the induction of the Src Family Kinase signalling cascade which mediates the migration of TLR9 from the ER to the late endosome, the uptake of free mCpGODN does not. However, it is suggested that encapsulation bypasses the methylation specific recognition of CpG ODN, allowing for the activation of SFK signalling resulting in subsequent co-localization of TLR9 and mCpG ODN in the endosome thus initiating immunostimulatory activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Liposomes are small phospholipid vesicles that have been widely investigated as drug carriers for the delivery of therapeutic agents. A variety of liposome formulations are presently under clinical trial exploration, while others have already been approved for clinical use. The aim of this study was to optimize liposome uptake into bacterial cells. Both gram-positive and gram-negative bacteria were used in the study as well as Candida albicans.Response surface methodology (RSM) using a central composite design (CCD) model was used to optimize liposomal formulations of carboxyfluorescien (CF) for each of the three microbes, and also the three microbes in combination namely; Staphylococcus aureus (Sa), Escherichia coli (Ec) and Candida albicans (Ca). Percentage of CF encapsulated and CF increase in Uptake were investigated with respect to two independent variables that were, cholesterol (CHOL) and stearylamine (SA) content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each microbe and the three microbes in combination. The model selected by the software managed to reasonably correlate the predicted models to the experimental data. Encapsulation of carboxyfluorescien (CF) into a liposome formulation enhanced its uptake by Staphylococcus aureus and Escherichia coli as well as Candida albicans. This was evident in the increase in CF uptake when the uptake rate of free CF was compared with that of liposomal CF.

Plant seeds, yeasts, bacterial spores, rotifers, and other organisms are capable of suspending their metabolism and entering a state of latency when dehydrated. These organisms may maintain this state for extremely long periods of time, yet upon rehydration resume normal metabolism, without evidence of severe membrane disruption. With many of these organisms, the ability to survive dehydration has been correlated to the production of large amounts of carbohydrates, including glycerol, glycogen and the disaccharide trehalose. Trehalose has been shown to protect isolated sarcoplasmic reticulum microsomes and phospholipid vesicles from dehydration damage, implying that the site of protective action of trehalose and other carbohydrates is the lipid portion of membranes. In this thesis, the effects of carbohydrate composition, vesicle size, and lipid composition on the protection of liposomes from dehydration was investigated, as was the structure of the solid lipid-trehalose complex. Electron microscopy of dried liposomes indicated that vesicles protected with trehalose remain essentially intact even when dry, while vesicles not protected by sugar are severely disrupted by drying . ³¹-P and ¹³-C NMR results suggested that the lipid of protected vesicles is in a similar phase as that of unprotected vesicles, and that this state is similar to powdered anhydrous phospholipid. Using carboxyfluorescein as a probe, it was demonstrated that trehalose, other sugars can prevent vesicle disruption upon dehydration. Different lipid compositions of the liposomes showed nearly identical behavior, with the exception of vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, which showed greater and lower stability to dehydration respectively. Light scattering experiments indicated that a wide variety of carbohydrate and lipid vesicle combinations can withstand dehydration and maintain their original size when protected by sugars. The implications of these results in the development of liposomes as pharmaceuticals are discussed, and a hypothesis is advanced regarding the role of carbohydrates in the preservation of dry lipid membranes. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Asthma is a chronic obstructive pulmonary disease (COPD) which affects 1 in every 12 Americans. Symptoms common to asthmatics include dyspnea, increased mucous production and airway hyperresponsiveness. While research over the past few decades has mostly established the immunological basis behind asthma, there have not been radical changes in the treatment modalities. It is believed that in many COPDs, alveolar macrophages play a critical role in disease progression. While evolutionarily, alveolar macrophages played a significant part in protecting the individual from harmful allergens, in asthma there may be an inappropriate activation of the alveolar macrophages to proteases such as cockroach allergen (CRA). Studies show that children living in inner cities with cockroach infestation are more likely to develop asthma than those that reside in rural areas with less exposure to cockroach allergens. In exposed individuals, when the alveolar macrophages come in contact with CRA, an immune cascade is initiated which sensitizes the child. Subsequent exposure to such an antigen will induce asthma like symptoms. One possible way of reducing such a response is to reduce the number of alveolar macrophages thus avoiding the pathalogical effects. Clodronate liposomes are liposomes that are encapsulated with bisphosphonate clodronate. When a macrophage phagocytoses such a liposome, the result is cellular suicide or apoptosis. In this study, we sensitized a murine model of CRA asthma and then monitored the impact of depleted alveolar macrophages using intratracheal administration of clodronate liposomes. We then studied the effect of this depletion on the recruitment of inflammatory cells such as neutrophils and eosinophils which are primary cellular contributors to the asthmatic response. Our studies show that while clodronate liposomes are effective in alveolar macrophage depletion, the subsequent inflammation through neutrophil recruitment interferes with the study of the delicate milieu of cells in the respiratory epithelium of this murine model.