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Receptor-mediated targeting of lipobeads bearing acetohydroxamic acid for eradication of Helicobacter pylori
Journal of Controlled Release 99 (2004) 27 – 40 www.elsevier.com/locate/jconrel
Receptor-mediated targeting of lipobeads bearing acetohydroxamic acid for eradication of Helicobacter pylori R.B. Umamaheshwari, N.K. Jain* Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar (M.P.) 470 003, India Received 25 October 2003; accepted 6 June 2004 Available online 10 August 2004
Abstract In the present context, phosphatidyl ethanolamine (PE) liposomes anchored polyvinyl alcohol (PVA) xerogel beads (lipobeads) bearing acetohydroxamic acid (AHA) was developed as a receptor-mediated drug delivery system for use in blocking adhesion of Helicobacter pylori and thereby preventing the sequelae of chronic gastric infections. PVA beads containing AHA were prepared by emulsification followed by low temperature crystallization method. Surface acylation with fatty acid chain was accomplished by treating PVA bare beads with palmitoyl chloride. The completion of this reaction was characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) which confirmed the formation of an ester bond. Final formation of lipobeads was accomplished by combining acylated PVA beads with a PE liposome suspension. To confirm the specific binding propensity of lipobeads towards the PE specific surface receptors of H. pylori, we have performed in situ adherence assay and radiolabelling assay with human stomach cells and KATO-III cells, respectively. In both of these studies, pretreatment of H. pylori with lipobeads completely inhibited the adhesion of H. pylori to human stomach cells and KATO-III cells. These assays could serve as suitable in-vitro models for the study of binding efficacy of lipobeads with H. pylori surface receptors. In addition, the antimicrobial activity of the formulations was evaluated by growth inhibition (GI) studies with isolated H. pylori strain. The inhibitory efficacy of lipobeads was significantly higher compared to that of PVA bare beads. These results suggest that lipobeads could be a potential targeted drug delivery system in the treatment of H. pylori. D 2004 Elsevier B.V. All rights reserved. Keywords: Receptor-mediated targeting; Helicobacter pylori; Phosphatidyl ethanolamine; Lipobeads; In situ adherence assay; Growth inhibition
1. Introduction
* Corresponding author. Tel.: +91-7582-2237-12; fax: +917582-2210-52. E-mail address: [email protected] (N.K. Jain). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.06.006
Helicobacter pylori is a gram-negative bacterium that is associated with the gastric inflammation, peptic ulcer, gastric cancer and is a risk factor for nonHodgkin’s lymphomas of the stomach [1]. It is a spiral shaped organism with a smoother outer coat with four
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to six bulbous tipped sheathed flagella at one end, which help to penetrate the mucosa and colonize on the surface of gastric antrum. Current evidences suggest that a number of virulence factors promote H. pylori colonization and infection at the antrum portion. Potential virulent determinants include motility [2], urease production [3], secretion of phospholipases [4], elaboration of cytotoxins [5] and adhesion [6]. An understanding of the strategies by which H. pylori persists in the gastric epithelium and the mechanisms underlying the host responses induced is thus of crucial importance to better understanding of the pathogenesis of H. pylori infections. Adherence of pathogenic bacteria to target cells is an important step in the pathogenesis of many bacterial diseases [7]. For example, following attachment of organisms to gut mucosal surfaces, host tissues are exposed to higher concentrations of bacterial enterotoxins. Adherence is also important for entry of organisms into epithelial cells. Histological examination of biopsy specimens from the antrum of human stomach has revealed the presence of H. pylori within gastric mucous and adherent to the apical membranes of gastric epithelial cells [8,9]. H. pylori was shown previously to bind to a specific alkylacyl glycerolipid derived from human erythrocytes, HEP2 cells and human antral epithelium [10]. Furthermore, cultured human cells with less PE show minimal attachment of H. pylori in vitro [11], emphasizing the importance of PE–H. pylori interaction in bacterial adhesion. PE is a predominant lipid in the antrum of the human stomach and functions as a receptor for H. pylori adhesion. Correlation of the ability of H. pylori to adhere to eukaryotic cells with the detected presence of the PE receptor, however, underscores the importance of this lipid as a major receptor in promoting H. pylori adhesion to intact cells. PE bacterial adhesin exists as a cell surfaceassociated ligand. On the basis of the above facts, antiadhesion drug delivery system based on PE has been developed as a receptor-mediated drug delivery system for use in blocking adhesion of Helicobacter and thereby preventing the sequelae of chronic gastric infections. A growing amount of literature describes the development and application of novel targeting and/ or release triggering schemes to improve the ther-
apeutic index of drugs encapsulated within PE liposomes [12]. However, phosphatidyl ethanolamine liposomes are unstable in acidic pH and in the presence of divalent cations; their loading capacity being limited by the water solubility of the material to be loaded [13]. Polymeric beads, although mechanically more stable and having a larger loading capacity than liposomes, lack many of the useful surface properties of a lipid bilayer shell. Jin et al. [14] described the preparation and characterization of a new hybrid vesicle system that combines complementary advantages of liposomes and polymeric beads. This system, which they have called lipobeads, consists of a lipid bilayer shell that is anchored on the surface of a hydrogel polymer core. It was therefore reasonable to expect that assembly of lipid bilayers on spherical hydrogel surfaces could be a useful approach for developing targeted drug delivery systems. We utilized this concept in developing targeted drug delivery systems based on PE in the treatment of H. pylori. Although H. pylori is susceptible to many antimicrobial agents, clinical trials with single antimicrobial agent(s) have not shown the complete eradication of H. pylori. This is because H. pylori exclusively resides on the luminal surface of the gastric mucosa under the mucous gel layer and access of antimicrobial drugs to the site of infection is restricted both from the stomach and from the gastric blood supply. For effective H. pylori eradication, therapeutic agent(s) must penetrate into the adhesion sites and permit localized release. With this optimistic approach, we have developed PE anchored PVA xerogel beads (lipobeads) bearing AHA in the treatment of H. pylori. Fig. 1 shows the schematic representation of the proposed theme. PE coating could plug and seal the receptors on H. pylori surface while continuous drug release occurs from lipobeads. Triple therapy consisting of combined use of antibiotics, such as amoxicillin and clarithromycin/ metronidazole, and a proton pump inhibitor, such as omeprazole, gives a high eradication rate, and is now applied for clinical treatment of H. pyloriassociated gastroduodenal disease. However, eradication is not always successful and harmful side effects of these drugs may be encountered. Moreover, the acquisition by H. pylori of resistance to
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2. Experiments 2.1. Materials PVA (degree of saponification: over 98.0 mol; mean degree of polymerization: 1000, 1700 and 3210) (almost hydrolyzed PVA), paraffin oil, Span 80, hexane and ethyl acetate were purchased from Merck (India). Palmitoyl chloride, phosphatidyl ethanolamine, cholesterol and oleic acid were purchased from Sigma (USA). Acetohydroxamic acid was purchased from Fluka Chemicals (USA). The other chemicals were of danalytical gradeT and doubledistilled water was used for all experiments. 2.2. Preparation and characterization of PVA beads
s
Fig. 1. Recognition of lipobeads by PE specific surface receptors of H. pylori.
antibiotics, including clarithromycin and metronidazole, has also become a serious problem that may disturb treatment efficacy. Therefore, non-antibiotic agents, which are both highly effective and safe, are required to diminish H. pylori-induced gastric lesions. Ohta et al. [15] evaluated the effects of urease inhibitors acetohydroxamic acid on H. pyloriinduced gastritis in Mongolian gerbils. Bacterial infection rates were reduced to 40–50% of the control values of 100% by the highest dose of AHA. Since antibiotic-resistant strains of H. pylori have become serious problem, non-antibiotic, urease inhibitors may be very useful to control H. pyloriassociated gastroduodenal disease. AHA is a small molecule (molecular weight 75.07), it can permeate intact bacterial cells and effectively inhibits the urease activity of H. pylori.
PVA beads were prepared by emulsification followed by low temperature crystallization method described previously [14]. PVA powder was introduced in hot water having concentrations of 5%, 10%, 15% and 22% w/w. Then AHA was added in PVA solutions at room temperature and then emulsified in paraffin oil (containing 1.5% w/v surfactant, Span 80) to form beads. The beads were then cross-linked by slow freezing the suspension at 20 8C at various times (24, 48, 96 and 192 h) followed by a slow thaw at 4 8C for 2 days. After freeze thawing, the oily portion was removed by extraction with ethanol, hexane and ethyl acetate. After extraction, the hot air was bubbled through the PVA bare beads suspension and the residual amount of moisture was removed from the PVA bare beads. After drying, they were stored in dessicator. AHA is soluble in ethanol so after extraction with ethanol of PVA bare beads, the free AHA was removed. The particle size and size distribution of PVA beads were determined in a Zetasizer 3000 HS (Malvern Instrument, UK). Surface morphology of PVA beads was determined by scanning electron microscopy (SEM, Jeol JX 840-A, Tokyo, Japan). A thin film of aqueous dispersion of PVA beads was applied on double stick tape over an aluminium stub and air-dried to get uniform layer of particles. These particles were coated with gold to a thickness of about 450 2 using Sputter gold coater. The samples were scanned and photographs were taken (Fig. 2).
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Fig. 2. SEM microphotograph of PVA hydrogel beads.
Drug entrapment efficiency was determined by dissolving PVA bare beads in hot aqueous solution and the digested homogenate was centrifuged at 3000 rpm for 3 min. The solution was filtered and the filtrate was assayed for AHA colorimetrically at 503.5 nm in a Shimadzu 1601 UV/Visible Spectrophotometer. 2.3. Preparation and characterization of acylated PVA beads Surface acylation with fatty acid chains was accomplished by treating the PVA beads with 1 M palmitoyl chloride in hexane at room temperature for 2–3 days and the solvent was evaporated. The palmitic species became anchored on the beads via esterification with surface hydroxyls. The completion of this reaction was characterized using ATR-FITR. For analyzing the surfaces with these analytical tools, PVA films were formed by identical chemical treatment as described above.
7.4 was used as aqueous medium (total lipid concentration, 84 mM) to hydrate the dry lipid film. Then they were sonicated for 20 min resulting liposomes were then evaluated for average vesicle size. Mean particle size and polydispersity index of PE liposomes were determined in a Zetasizer (Malvern Instrument). Final formation of lipobeads was accomplished by combining equal parts of a suspension of surface modified beads with a PE liposomes suspension. Beads were added to the PE liposome dispersions with vortexing for 2 min in microfuge tubes. In this manner, the PE liposomes are reported to spontaneously collapse into a continuous bilayer surrounding the beads. After 10 min, the beads were then centrifuged and resuspended in buffer, repeating five times to remove unbound liposomes and free lipids, thus leaving single bilayer covered beads. Size distribution of lipobeads was measured in a Zetasizer (Malvern Instrument). To investigate the homogeneous distribution of liposomal bilayer on the PVA bare beads, confocal microscopy study was carried out. Rhodamine 123-PE liposomes were prepared by lipid cast film hydration method [16]. Rhodamine 123 bearing lipobeads were prepared by mixing equal parts of a suspension of surface modified beads with rhodamine 123-PE liposome suspension. Sample was viewed and imaged using a Leica LSM 510 system equipped with an argon ion laser. Additional excitation sources included HeNe1 and HeNe2 lasers for the respective 538 and 633 nm excitation wavelengths were performed at room temperature (~20 8C). Imaging experiments were typically performed using a 263/1.3 oil immersion objective with the diaphragms set to allow the probing of ~1 Am wide vertical slices of the imaged beads.
2.4. Preparation of lipobeads 2.5. Drug release studies PE liposomes were prepared by lipid cast film hydration method [16]; lipids, phosphatidyl ethanolamine (PE), oleic acid and cholesterol were dissolved in minimum quantity of chloroform in a round-bottom flask. A thin film of the lipids was cast on the inner surface of the round-bottom flask by evaporating the solvent under reduced pressure using rotary evaporator (Buchi type, York Sci., Mumbai). The flask was constantly rotated until dried film was formed. Final traces of solvent were removed under vacuum. 10 mM HEPES buffer containing 0.8% NaCl, adjusted to pH
The effect of PVA concentration, freezing time and PE bilayer on AHA release rate was observed. The drug release studies were carried out by USP dissolution apparatus paddle type at 37F1 8C. The dissolution fluid (400 ml) was simulated gastric fluid (SGF, pH 1.2). The PVA bare beads and lipobeads were held at the bottom of the vessel in a stainless-steel wire mesh (sinker). The paddle was positioned approximately 2.5 cm from the bottom of the vessel and was rotated at 50 rpm. An aliquot (2 ml) of
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dissolution fluid was removed at predetermined times and 2 ml of fresh fluid was added to the vessel to maintain the original volume. The concentrations of AHA were analyzed colorimetrically at 503.5 nm by using Shimadzu 1601 UV/Visible spectrophotometer. 2.6. Agglutination assay The bacterial strains H. pylori 1101 (isolated from a patient suffering from functional dyspepsia and shown to have erosive gastritis upon endoscopy) and H. pylori 69A (isolated from a patient with non-ulcer dyspepsia) were used in this study. H. pylori was grown on agar plates made of brain heart infusion agar containing 0.25% yeast extract, 10% sheep blood and supplemented with 0.4% Campylobacter selective complement (Skirrrow supplement, SR 69), maintained in a micro-aerophillic atmosphere (85% N2, 10% CO2, 5% O2) for 2 days, harvested under sterile conditions into brain heart infusion agar and finally diluted in Dulbecco’s modified Eagle’s medium plus Ham’s F12 (DMEM Ham’s 1:1) (pH 5.4) without antibiotics at 108 bacteria ml1. A bacterial cell suspension of H. pylori (2108, colony-forming units (CFU)/ml) in phosphate-buffered saline (PBS, pH 7.4) was prepared. This bacterial suspension was mixed with one-tenth of its volume of the lipobeads suspension, commercial PE and PVA bare bead suspension in a small glass test tube. The mixture was then incubated at 37 8C with or without shaking. 2.7. Adherence assay (radiolabelling assay) 14
C-labelled (1.7 ACi/ml) proline and histidine were added to the growth medium at the time of inoculation with bacteria. H. pylori strain 69A was used for all radiolabelling studies as described previously [17]. Incubation conditions were identical to those described above. Since 14C-labelled proline and histidine were used, gases which were released into the culture jar were absorbed in Protosol. After incubation for 12 h, bacteria were pelleted by centrifugation at 3000 rpm for 15 min, washed three times in sterile PBS (pH 7.4) and resuspended in PBS (pH 7.4) to an optical density of 0.5 at 600 nm. Incorporation of radioactive label into the bacteria was determined by counting disintegrations per min (dpm) in a h-scintillation counter.
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KATO-III cells obtained from the American Type Culture Collection (Rockville, MD) are gastric epithelial cells originally derived from a human gastric adenocarcinoma. The cells were grown in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum 20% v/v, penicillin 100 IU/ml and streptomycin 0.1 mg/ml. Cells were cultured in polystyrene tissue culture flasks in an atmosphere of 5% CO2 in air and 98% humidity at 37F1 8C. Confluent growth was obtained when flasks were incubated for 8–10 days, changing the growth medium at least once every 3 days. Cells were counted on a haemocytometer slide by phase-contrast microscopy; 104 cells/ml was initially attached to the polystyrene and 103 cells/ml remained in the suspension. KATO-III cells were subcultured and grown in 12well tissue-culture cluster dishes. After 8–10 days, the number of gastric epithelial cells adherent to polystyrene was approximately 3106 cells/well. H. pylori cells grown in supplemented Brucella broth for 14 h were resuspended in RPMI-1640 medium without antibiotics. The following suspensions were prepared, H. pylori incubated (1 h) with lipobeads (HP-LB), H. pylori incubated (1 h) with PVA bare beads (HP-PB) and H. pylori alone (HP). H. pylori suspensions were washed once in PBS (pH 7.4) and were added to KATO-III cells. For quantitation of H. pylori adherence to KATOIII cells, 108 3H-labelled bacteria were resuspended in 1 ml of RPMI-1640 medium, without antibiotics and added to the wells of a cluster dish. The cluster dishes were incubated in micro-aerophilic conditions for 3.5 h at 37F1 8C. Non-adherent bacteria were then removed by six washes with 1 ml of PBS (pH 7.4). To remove adherent H. pylori and KATO-III cells from the wells, trypsin 0.25% w/v was added and incubated at 37F1 8C for 10 min. The number of bacteria adherent to KATO-III cells and polystyrene surfaces was determined by counting dpm in 0.1 ml samples collected from each well after trypsination. Viability of KATOIII cells was determined at the beginning and end of the assays by trypan blue exclusion. 2.8. In situ adherence assay with human stomach cells Multiple samples of adult human oesophagus, stomach, duodenum and colon were obtained from the surgical pathology and autopsy files of the Dr.
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Periyar Government hospital, Madurai, India. Only samples of nondiseased tissues were used in these experiments. All tissues were fixed in 10% formalin or in a solution of picric acid/formaldehyde/glacial acetic acid (15:5:1, Bouin’s fluid) and subsequently embedded in paraffin [18]. Five micron sections were prepared and used for haematoxylin–eosin staining (to identify cell types present in gastric units and to verify that the tissue samples had no pathologic changes) and/or for subsequent adherence assays. Tissue sections were deparaffinized in xylene and isopropanol, rinsed in water followed by PBS (pH 7.4) and then incubated for 15–30 min in blocking buffer (0.2% bovine serum albumin/0.05% Tween 20, prepared in PBS (pH 7.4). The FITC-labeled bacterial suspension was diluted 20-fold in blocking buffers and 200 Al was placed on the slide, which was then incubated for 1 h at room temperature in a humidified chamber. Slides were subsequently washed four to six times with PBS (pH 7.4) prior to inspection. To analyze the ability of formulations to block binding, 200 Al suspensions of FITC-labeled bacteria were preincubated for 2 h at room temperature with lipobeads and PVA bare beads. Bacteria were washed once in blocking buffer before the mixture was added to tissue sections. After incubation, the in situ binding was observed by using fluorescence microscopy (Leica, Germany). 2.9. In vitro growth inhibition (GI) studies In vitro GI studies were preformed with H. pylori strains NCTC 11637. Percentage bacterial growth inhibition (%GI) was the parameter studied. H. pylori
was grown on agar plates made of brain heart infusion agar containing 0.25% yeast extract, 10% sheep blood and supplemented with 0.4% Campylobacter selective complement (Skirrrow supplement, SR 69), maintained in a micro-aerophillic atmosphere (85% N2, 10% CO2, 5% O2) for 2 days, harvested under sterile conditions into brain heart infusion agar and finally diluted in PBS (pH 7.4) without antibiotics at 108 bacteria ml1. The bacterial suspensions were centrifuged at 600 rpm for 30 min. These experiments were repeated thrice and the bacterial cell concentration approximately adjusted by dilution with PBS (pH 7.4) for measuring the absorbance at 660 nm. The following formulations bearing AHA (twice the minimum inhibitory concentration (MIC)—7 mM) were added to the H. pylori suspension to a final volume of 10 ml per sterile tube. 1. 2. 3. 4. 5.
AHA bearing PVA bare beads—PVA bare beads AHA bearing lipobeads—Lipobeads Drug free PVA bare beads—Placebo I Drug free lipobeads—Placebo II Plain AHA—AHA
The tubes were incubated with constant agitation at 37F1 8C under micro-aerophilic conditions. In order to check any antimicrobial activity of constitutive ingredients, the drug free PVA bare beads (Placebo I) and lipobeads (Placebo II) were taken for the studies. The bacterial growth was monitored spectrophotometrically at 660 nm in a Shimadzu 1601 UV/Visible spectrophotometer against blank (uninoculated broth) at 6, 12 and 24 h. This procedure was repeated thrice.
3. Results and discussion Lipobeads may potentially be useful in several novel biomedical applications. As a drug carrier, lipobeads should be superior to liposomes in the sense of enhanced mechanical stability. Long-term storage is possible since acylated beads loaded with drug can be stored in a dry form before lipid coating. Also, many of the techniques developed for liposomes such as stimuli-sensitive drug release, steric stabilization and receptor- or antibodymediated drug targeting [19] should still be applicable with lipobeads. As with other human pathogens, it has been proposed that H. pylori species express multiple adhesins that would allow bacterial interaction with various target cell epitopes on mucin components including fucosylated glycoconjugates and phosphatidyl ethanolamine [11]. For effective H. pylori eradication, the drug delivery systems must plug and seal the H. pylori as well as continuously release the antibiotics at the infection site in effective concentration and in fully active form.
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Development of PE liposomes in H. pylori treatment has many challenges. PE constitutes one such class of lipids, which when dispersed in pure form under physiological conditions assemble into nonbilayer structures. This is in contrast to lamellar phase forming lipids, which readily form hydrated lamellar phase or bilayer structures when dispersed in aqueous media [20]. We developed liposomes anchored PVA hydrogel beads to overcome the limitations of the PE liposomes in H. pylori treatment as described previously [14]. One approach has been to create a hydrophobic anchor for the bilayer by attaching lipid molecules to the surface of preformed PVA xerogel (dry hydrogel) beads. When the surface modified xerogel is hydrated and treated with a PE liposomes suspension, the hydrophobic lipid molecules and other intrinsic membrane components of the PE liposomes associate spontaneously with the hydrophobic fatty acid anchors on the surface of the polymer and self-organize into a membrane distributed over that surface through hydrophobic interactions. 3.1. Preparation and characterization of PVA beads PVA beads were prepared by emulsification followed by low temperature crystallization method and this procedure produced PVA xerogel spheres ranging from 50 to 100 Am Maximum size distribution of lipobead was 75F10 Am. Spherical beads ranging in size from 80 to 100 Am were selected by sieving for further modification. Lipobead yield after sieving was 68.3%. The surface morphology of PVA hydrogel beads was investigated by scanning electron microscopy that revealed the spherical shape with smooth surface (Fig. 2). The drug entrapment efficiency was increased with PVA concentrations. At 22% w/w PVA concentration, the entrapment efficiency (65.3%) was high when compared to 5%, 10% and 15% w/w concentration. 3.2. Preparation and characterization of acylated PVA beads Surface acylation with fatty acid chain was accomplished by treating PVA bare beads with 1 M palmitoyl chloride in hexane at room temperature for 2–3 days. The completion of reaction was characterized by using attenuated total reflectance ATR-FTIR spectroscopy study. In brief, the infrared beam enters the ATR crystal from one of the side faces. If the refractive index of the crystal is higher than the PVA, and the incident angle of the beam is higher than a critical angle, then the infrared beam is totally reflected at the crystal/PVA interface, and the beam travels inside the crystal and exits from the other side face. However, due to diffraction at the crystal surface, a small fraction of the beam penetrates into the PVA layer and is absorbed by PVA. The fraction of the beam which is absorbed gives rise to absorption bands in the ATR spectrum and is used to monitor the concentration of each component within the penetration depth in the polymer cage. In ATR-FTIR spectrum of palmitoyl chloride anchored PVA, the band at 1776 cm1 was for ester group which was formed between the carboxyl group of palmitoyl chloride and the hydroxyl group of PVA (data not shown). The formation of an ester bond was determined and 53% changes in atomic ratio at the surface were measured by ATR-FTIR. 3.3. Preparation and characterization of lipobeads PE liposomes prepared were studied by light microscopy and transmission electron microscopy. The electron microphotographs showed that liposomes were unilamellar vesicles and composed of bilayer unit membranes, about 75 2 thick. Mean diameter of PE liposome formulation was 4.2F1.2 Am. Final formation of lipobeads was accomplished by combining PVA bead suspension to a PE liposome suspension. Anchoring of PE liposomes on PVA hydrogel beads was confirmed by swelling studies. When dried acylated beads are placed in water, their diameter increases about one to four times at swelling equilibrium. However, the beads aggregate and float on the water surface due to their surface hydrophobicity. Adding a liposome suspension to the solution with floating beads causes the beads to separate and sink to the bottom.
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We measured contact angle of PVA bare beads, acylated PVA beads and lipobeads. For acylated PVA surfaces, the contact angle was 1058 indicating that the surface is hydrophobic. The contact angle of the surface was reduced to less than 158 after the film was treated with liposomes. When beads are separated from water by filtration and exposed to air, the bilayer is disturbed and the beads again became hydrophobic. The contact angle of the beads again became N1008. Fig. 3 shows a confocal fluorescence microphotograph of a homogeneous lipid bilayer surrounding a lipobead when viewed as a 1 Am slice at the focal depth at the equator of a bead. From this photograph, we concluded the homogeneous distribution of bilayer on the PVA bare beads. An advantage of using beads as a platform for PE bilayers supported assemblies is the direct exposure of the number of PE sites that are readily afforded by H. pylori receptors. In these experiments, varying the mole fraction of PE that is mixed with oleic acid and cholesterol regulates the number of PE molecules per bead. Thus bead supported bilayers derived from a mixture of PE, oleic acid and cholesterol of known stoichiometry can result in a bead population with a reasonably defined surface coverage of PE. This is established by dividing the surface area of the bead (4pr 2) by the area of the average lipid head group. The desired surface coverage of PE is then simply affected by using appropriate mole fraction of PE in the vesicle preparation. By extension of this approach, one can also ensure that an excess amount of lipids is always mixed with beads. 3.4. Drug release studies In vitro release studies of AHA were carried out with PVA bare beads which were prepared at various freezing times and PVA concentrations. Fig. 4A shows the release profiles of AHA from PVA bare beads (22% w/w PVA, pH 1.2), which were prepared at various freezing times at 20 8C. The release rate of AHA was slower on longer freezing times. Fig. 4B shows the effect of PVA concentrations at pH 1.2 on the release profiles of AHA. Slower release rate of AHA was observed at 22% w/w concentrations of PVA. To investigate the effect of PE bilayer on in vitro drug release rate of AHA, both lipobeads and PVA bare beads were taken. Fig. 4C shows the effect of bilayer on the release profile of AHA from PVA bare beads. AHA was released at higher rate from PVA bare beads than lipobeads. From Fig. 4C, it was concluded that PE bilayer significantly slower/control the AHA release rate. Till 6 h, the release rate was significantly lower with lipobeads when compared to PVA bare beads. After 6 h, difference between the release rate of PVA beads and lipobeads was decreased. Due to the hydrophilicity of the PVA bare beads,
Fig. 3. Confocal fluorescence photomicrograph of lipobeads.
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Fig. 4. Release profiles of AHA from PVA bare beads at various (A) freezing times (PVA concentration 22% w/w) and (B) concentrations of PVA (freezing time 48 h). (C) Effect of PE bilayer on release rate of AHA from PVA bare beads (PVA concentration 22% w/w).
the drug release rate was higher whereas due to the hydrophobicity of lipobead formulations, the drug release rate was slower up to 6 h. Similar release rate of PVA bare beads and lipobeads might be due to the reduction in hydrophobicity of lipobeads after 6 h. Based on in vitro drug release studies and drug entrapment efficiency, PVA bare beads prepared at 22% w/w PVA concentration (freezing time 48 h) were taken for further studies.
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3.5. Agglutination assay H. pylori showed different agglutination reactions with various formulations. Among the three formulations tested—lipobeads, PVA bare beads and commercial PE—strong agglutinations were observed with the specific lipobeads and commercial PE (Table 1). All H. pylori organisms were agglutinated by the lipobeads whereas PVA bare beads were found to be non-reactive with H. pylori strains. The agglutinating ability of lipobeads towards H. pylori might be due to the retention of PE specific receptors on H. pylori surface. It is interesting to note that PE on the lipobeads as well as commercial PE was recognized by all H. pylori strains. Lipobeads showed the same binding characteristics with PE specific surface receptors of H. pylori as the commercial PE. It revealed the intact specificity of homogeneous distribution of PE liposomes on the PVA bare beads. It further confirmed the previous report that PE is more likely to be function as a receptor for H. pylori related adhesion [11]. 3.6. Adherence assay (radiolabelling assay) In the present study, we have demonstrated the attachment of H. pylori to epithelial cells of gastric origin in vitro. Adherence of H. pylori to KATO-III cells was morphologically identical to the in vivo observations reported previously [17]. In the present bacterial adherence assays, the number of attached labeled bacteria was determined by measurement of radioactivity. As H. pylori requires incubation for z5 days to achieve adequate growth on agar media, a rapid and reproducible method for quantitation of bacterial adherence with radiolabelled organisms was employed in this study as described previously [17]. Minimal binding of radiolabelled bacteria to plastic alone and the negligible loss of radioisotope from H. pylori both indicated that free radioisotope was not an important confounding variable in these experiments. Bound radioactivity of HP and HP-PB with KATO-III cells was greater than HP-LB and the number of H. pylori adhering to KATO-III cells was similar with HP and HP-PB (Fig. 5). Adherence of H. pylori to tissue culture cells was significantly reduced with HP-LB, because PE receptors of organisms were completely plugged and sealed by the HP-LB formulations. The reduction in bacterial binding to KATO-III cells with HP-LB was probably related to less availability of PE receptor as previously observed by Bitzan et al. [21]. They showed that bovine colostrum completely blocked the bacterial attachment to PE, Gg4 and Gg3. Colostral lipid extracts of bovine colostrum contained PE and lyso-PE that bound H. pylori in vitro. Similarly, lipobeads completely plug and seal the PE receptor and block the H. pylori adhesion with KATO-III cells. 3.7. In situ adherence assay with human stomach cells In situ adherence assay should be useful in further characterizing the H. pylori adhesin receptors and for identifying therapeutically useful compounds that inhibit strain specific and cell-lineage specific binding of this human pathogen. Falk et al. [22] developed this experiment to characterize the surface receptors present on H. pylori. We followed the same reported and validated protocol for in situ adherence assay to evaluate the targeting propensity of lipobead formulation. The drug-free formulations, i.e. lipobeads and PVA bare beads, were incubated with H. pylori strains for 2 h. The following three samples were overlaid on sections of Table 1 Lipobeads agglutination patterns of H. pylori (n=3) Formulations
H. pylori strain 69A
H. pylori strain 1101
Lipobeads PVA bare beads Commercial PE
+++ +++
+++ +++
The agglutination reaction was scored as follows: +++ = strong positive reaction with large clumps; ++ = strong-positive reaction with moderate size clumps; + = positive agglutination with fine clumps; w = weak agglutination; = no agglutination.
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Fig. 5. Radioactivity of adherent HP-LB, HP-PB and HP with KATO-III cells. HP-LB- H. pylori suspension preincubated with lipobeads. HPPB- H. pylori suspension preincubated with PVA bare beads. HP – H. pylori suspension.
human stomach cells: H. pylori incubated with lipobeads (HP-LB), H. pylori alone (HP) and H. pylori incubated with PVA bare beads (HP-PB). The fluorescence microphotographs reveal the binding intensity of H. pylori samples with human gastric mucosa. Fig. 6A indicates that HP was strongly bound at surface mucous cells situated in the human stomach portion. The intensity of binding was significantly higher in HP compared to that of HP-LB (Fig. 6A and C). HPPB showed equal intensity of binding like H. pylori with the human stomach cells (Fig. 6B). The intensity of binding was almost nil in HP-LB (Fig. 6C). The results clearly revealed that the PE receptors were completely bound with the lipobeads. Therefore HP-LB strain could not bind with the human stomach cells. PE specific receptors are intact in the case of HP-PB and HP suspensions, thus they strongly bind with the human stomach cells. In situ adherence assay confirmed the superior binding propensity of lipobeads with PE specific surface receptors of H. pylori. 3.8. In vitro growth inhibition studies In the present study, lipobeads–H. pylori interaction and adsorption to the bacterial cell surface could be exploited for effective localization and targeting of lipobeads to a preselected bacterial cell line. The method selected for the proposed study was turbidometry based on optical density measurements [18]. Quantitation of lipobeads– bacterial interaction was approached in terms of percentage growth inhibition (%GI). One of the major advantages of culture is that it allows sensitivity testing of H. pylori to the antimicrobial agents used in the treatment. The effect of different drug-loaded and drug-free formulations on bacterial growth was investigated. It was reported that AHA specifically inhibits the urease enzyme, which is secreted by the H. pylori [23]. The antimicrobial effect of formulations was determined in terms of percentage growth inhibition (%GI) that was calculated as the ratio of optimal density (OD) (at 660 nm) of a given mixture against that of tubes containing H. pylori alone and can be expressed as given below: %GI ¼
OD of test organism at a particular time interval OD of test mixture at the same time interval 100 OD of test organism at a particular time interval
All of these results of these experiments are present in Fig. 7. The results could be arranged to an order of %GI performances: Lipobeads N AHA N PVA bare beads N PlaceboI N PlaceboII
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Fig. 6. Section of human adult stomach incubated with FITC-labeled (A) HP (H. pylori alone), (B) HP-PB (H. pylori incubated with lipobeads) and (C) HP-LB (H. pylori incubated with PVA bare beads) (450).
Placebo I (drug-free PVA bare beads) and Placebo II (drug-free lipobeads) exhibited insignificant growth inhibition (Fig. 7), which suggested that constitutive ingredients have no antimicrobial activity. They can only interact with the bacterial cell surfaces like cell adhesion of PE and interaction of PVA hydrogel conjugates on the tight junction of epithelium cells. Both PVA bare beads and lipobeads have the drug concentration equivalent to MIC (3.5 mM). After 6 h, %GI values were 35F4%, 25F4% and 100% for AHA, PVA bare beads and lipobeads, respectively. The bacterial growth was inhibited remarkably by lipobeads than PVA bare beads and drug (AHA). Lipobeads showed superior %GI than the equal amount of drug bearing PVA bare beads and AHA (Fig. 7). It could be assumed that the affinity of lipobeads towards phosphatidyl ethanolamine specific receptors present on H. pylori surface might be instrumental in pursuing better results.
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Fig. 7. Percentage growth inhibition of formulations.
The %GI values of formulations at different time intervals are shown in Fig. 7. The PVA bare beads completely inhibit the bacterial growth only after 24 h. Although the drug release rate of lipobeads was slower than PVA bare beads (Fig. 4C), lipobeads were completely inhibiting the H. pylori growth within 6 h. Thus, the %GI of lipobeads was independent of drug concentration and incubation time. This could be due to the fact that drug targeting was predominate as a result of the specificity of the lipobeads towards the PE specific receptors on the bacterial surface glycocalyx. PE–H. pylori interaction could lead to increase in drug concentration as well as therapeutic index at the H. pylori surface.
4. Conclusions
Acknowledgements
The characterization studies suggested that lipobeads could be a novel delivery device to improve the bioavailability of anti-H. pylori agents which are aimed to produce a local and specific effect in the stomach and are specifically absorbed through the upper region of the stomach. The plugging and sealing effect of the lipobeads could also impart superior targetability on the H. pylori surface. The proposed site specific drug delivery system targets the H. pylori more effectively and could serve to optimize antibiotic monotherapy of H. pylori based infections. These systems could protect the drug from the gastric environments. From all of the experiments performed, it can be suggested that the developed lipobeads could be successful in the treatment of H. pylori. In vivo growth inhibition studies of drug bearing lipobeads with animal models in order to evaluate the efficiency of formulations in vivo may further substantiate the present conclusions.
Authors would like to acknowledge Dr. Yegneswaran, Deputy Director, Regional Research Laboratory, Bhopal, India and Director, Madurai Kamarajar University, Madurai, India for providing necessary facilities. R.B. Umamaheshwari acknowledges Indian Council of Medical Research, New Delhi, India for awarding Senior Research Fellowship during the tenure of this work.
References [1] G. Bode, P. Malfertheiner, H. Ditschuneit, Pathogenetic implications of ultrastructural findings in Campylobacter pylori related gastroduodenal diseases, Scand. J. Gastroenterol. 23 (1988) 25 – 39. [2] K.A. Eaton, D.R. Morgan, S. Krakowka, Virulence factors for Campylobacter pylori in gnotobiotic piglets, Infect. Immun. 57 (1989) 1119 – 1125. [3] S.L. Hazell, A. Lee, Campylobacter pylori, urease, hydrogen ion back diffusion, and gastric ulcers, Lancet ii (1986) 15 – 17.
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[4] N. Dorrell, M.C. Martino, R.A. Stabler, S.J. Ward, Z.W. Zhang, A. McColm, J.M.G. Farthing, B.W. Wren, Characterization of Helicobacter pylori PLdA, a phospholipase with a role in colonization of the gastric mucosa, Gastroenterology 117 (1999) 1098 – 1104. [5] R.D. Leunk, P.T. Johnson, B.C. David, W.C. Kraft, D.R. Moryan, Cytotoxic activity in broth-culture filtrates of Campylobacter pylori, J. Med. Microbiol. 26 (1988) 93 – 100. [6] C.A. Lingwood, G. Wasfy, H. Han, M. Huesca, Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori, Infect. Immun. 61 (1993) 2474 – 2478. [7] R.P. Logan, Adherence of Helicobacter pylori, Aliment. Pharmacol. Ther. 10 (1996) 3 – 15. [8] G. Bukholm, T. Tannaes, P. Nedenskor, Y. Esbensen, H.J. Grar, T. Herig, S. Ariansen, I. Guldrog, Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition, Scand. J. Gastroenterol. 32 (1997) 445 – 454. [9] B.D. Gold, M. Huesca, P.M. Sherman, C.A. Lingwood, Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro, Infect. Immun. 61 (1993) 2632 – 2638. [10] I. Corthesy-Theulaz, N. Porta, E. Pringault, Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers is pH dependent, Infect. Immun. 64 (1996) 3827 – 3832. [11] C.A. Lingwood, M. Huessca, A. Kuksis, The glycerolipid receptor for Helicobacter pylori (and Exoenzyme S) is phosphatidyl ethanolamine, Infect. Immun. 60 (1992) 2470 – 2476. [12] V.O. Gerasimov, A.J. Boomer, M.M. Qualls, D.H. Thompson, Cytosolic drug delivery using pH- and light sensitive liposomes, Adv. Drug Deliv. Rev. 38 (1999) 317 – 318. [13] N. Duzgunes, P.M. Straubinger, P.A. Baldwin, D.S. Friend, D. Papahadjopoulas, Proton-induced fusion of oleic acid–phosphatidyl ethanolamine liposomes, Biochemistry 24 (1985) 3091 – 3098.
[14] T. Jin, P. Pennefather, P.I. Lee, Lipobeads: a hydrogel anchored lipid vesicle system, FEBS Lett. 397 (1996) 70 – 74. [15] T. Ohta, H. Shibata, T. Kawamori, M. Iimuro, T. Sugimura, K. Wakabayashi, Marked reduction of Helicobacter pyloriinduced gastritis by urease inhibitors, acetohydroxamic acid and fluorafamide, in Mongolian gerbils, Biochem. Biophys. Res. Commun. 285 (2001) 728 – 733. [16] V.P. Torchilin, A.N. Lukyyanor, A.L. Klibnor, V.G. Omelyanenk, Interaction between oleic acid-containing pH-sensitive and plain liposomes fluorescent microscopy studies, FEBS Lett. 305 (1992) 185 – 188. [17] S.G. Hemalatha, B. Drumm, P. Sherman, Adherence of Helicobacter pylori to human gastric epithelial cells in vitro, J. Med. Microbiol. 35 (1991) 197 – 202. [18] R.B. Umamaheshwari, P. Jain, N.K. Jain, Site-specific delivery of acetohydroxamic acid in the treatment of H. pylori, STP Pharma Sci. 13 (2003) 41 – 48. [19] G. Gregordiasis, Liposome Technology, 2nd ed., vol. III, CRC Press, Boca Raton, FL, 1994. [20] I.M. Hafez, P.R. Cullis, Cholesteryl hemisuccinate exhibits pH sensitive polymorphic phase behavior, Biochim. Biophys. Acta 1463 (2000) 107 – 114. [21] M.M. Bitzan, B.D. Gold, D.J. Philpott, Inhibition of Helicobacter pylori and Helicobacter mustelae binding to lipid receptors by bovine colostrum, J. Infect. Dis. 177 (1998) 955 – 961. [22] P. Falk, K.A. Roth, T.T. Boren, G. Westbolm, J.I. Gordon, An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 2035 – 2039. [23] R.B. Umamaheshwari, S. Jain, D. Bhadra, N.K. Jain, Floating microspheres bearing acetohydroxamic acid for the treatment of Helicobacter pylori, J. Pharm. Pharmacol. 55 (2004) 1607 – 1613.
Receptor-mediated targeting of lipobeads bearing acetohydroxamic acid for eradication of Helicobacter pylori R.B. Umamaheshwari, N.K. Jain* Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar (M.P.) 470 003, India Received 25 October 2003; accepted 6 June 2004 Available online 10 August 2004
Abstract In the present context, phosphatidyl ethanolamine (PE) liposomes anchored polyvinyl alcohol (PVA) xerogel beads (lipobeads) bearing acetohydroxamic acid (AHA) was developed as a receptor-mediated drug delivery system for use in blocking adhesion of Helicobacter pylori and thereby preventing the sequelae of chronic gastric infections. PVA beads containing AHA were prepared by emulsification followed by low temperature crystallization method. Surface acylation with fatty acid chain was accomplished by treating PVA bare beads with palmitoyl chloride. The completion of this reaction was characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) which confirmed the formation of an ester bond. Final formation of lipobeads was accomplished by combining acylated PVA beads with a PE liposome suspension. To confirm the specific binding propensity of lipobeads towards the PE specific surface receptors of H. pylori, we have performed in situ adherence assay and radiolabelling assay with human stomach cells and KATO-III cells, respectively. In both of these studies, pretreatment of H. pylori with lipobeads completely inhibited the adhesion of H. pylori to human stomach cells and KATO-III cells. These assays could serve as suitable in-vitro models for the study of binding efficacy of lipobeads with H. pylori surface receptors. In addition, the antimicrobial activity of the formulations was evaluated by growth inhibition (GI) studies with isolated H. pylori strain. The inhibitory efficacy of lipobeads was significantly higher compared to that of PVA bare beads. These results suggest that lipobeads could be a potential targeted drug delivery system in the treatment of H. pylori. D 2004 Elsevier B.V. All rights reserved. Keywords: Receptor-mediated targeting; Helicobacter pylori; Phosphatidyl ethanolamine; Lipobeads; In situ adherence assay; Growth inhibition
1. Introduction
* Corresponding author. Tel.: +91-7582-2237-12; fax: +917582-2210-52. E-mail address: [email protected] (N.K. Jain). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.06.006
Helicobacter pylori is a gram-negative bacterium that is associated with the gastric inflammation, peptic ulcer, gastric cancer and is a risk factor for nonHodgkin’s lymphomas of the stomach [1]. It is a spiral shaped organism with a smoother outer coat with four
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to six bulbous tipped sheathed flagella at one end, which help to penetrate the mucosa and colonize on the surface of gastric antrum. Current evidences suggest that a number of virulence factors promote H. pylori colonization and infection at the antrum portion. Potential virulent determinants include motility [2], urease production [3], secretion of phospholipases [4], elaboration of cytotoxins [5] and adhesion [6]. An understanding of the strategies by which H. pylori persists in the gastric epithelium and the mechanisms underlying the host responses induced is thus of crucial importance to better understanding of the pathogenesis of H. pylori infections. Adherence of pathogenic bacteria to target cells is an important step in the pathogenesis of many bacterial diseases [7]. For example, following attachment of organisms to gut mucosal surfaces, host tissues are exposed to higher concentrations of bacterial enterotoxins. Adherence is also important for entry of organisms into epithelial cells. Histological examination of biopsy specimens from the antrum of human stomach has revealed the presence of H. pylori within gastric mucous and adherent to the apical membranes of gastric epithelial cells [8,9]. H. pylori was shown previously to bind to a specific alkylacyl glycerolipid derived from human erythrocytes, HEP2 cells and human antral epithelium [10]. Furthermore, cultured human cells with less PE show minimal attachment of H. pylori in vitro [11], emphasizing the importance of PE–H. pylori interaction in bacterial adhesion. PE is a predominant lipid in the antrum of the human stomach and functions as a receptor for H. pylori adhesion. Correlation of the ability of H. pylori to adhere to eukaryotic cells with the detected presence of the PE receptor, however, underscores the importance of this lipid as a major receptor in promoting H. pylori adhesion to intact cells. PE bacterial adhesin exists as a cell surfaceassociated ligand. On the basis of the above facts, antiadhesion drug delivery system based on PE has been developed as a receptor-mediated drug delivery system for use in blocking adhesion of Helicobacter and thereby preventing the sequelae of chronic gastric infections. A growing amount of literature describes the development and application of novel targeting and/ or release triggering schemes to improve the ther-
apeutic index of drugs encapsulated within PE liposomes [12]. However, phosphatidyl ethanolamine liposomes are unstable in acidic pH and in the presence of divalent cations; their loading capacity being limited by the water solubility of the material to be loaded [13]. Polymeric beads, although mechanically more stable and having a larger loading capacity than liposomes, lack many of the useful surface properties of a lipid bilayer shell. Jin et al. [14] described the preparation and characterization of a new hybrid vesicle system that combines complementary advantages of liposomes and polymeric beads. This system, which they have called lipobeads, consists of a lipid bilayer shell that is anchored on the surface of a hydrogel polymer core. It was therefore reasonable to expect that assembly of lipid bilayers on spherical hydrogel surfaces could be a useful approach for developing targeted drug delivery systems. We utilized this concept in developing targeted drug delivery systems based on PE in the treatment of H. pylori. Although H. pylori is susceptible to many antimicrobial agents, clinical trials with single antimicrobial agent(s) have not shown the complete eradication of H. pylori. This is because H. pylori exclusively resides on the luminal surface of the gastric mucosa under the mucous gel layer and access of antimicrobial drugs to the site of infection is restricted both from the stomach and from the gastric blood supply. For effective H. pylori eradication, therapeutic agent(s) must penetrate into the adhesion sites and permit localized release. With this optimistic approach, we have developed PE anchored PVA xerogel beads (lipobeads) bearing AHA in the treatment of H. pylori. Fig. 1 shows the schematic representation of the proposed theme. PE coating could plug and seal the receptors on H. pylori surface while continuous drug release occurs from lipobeads. Triple therapy consisting of combined use of antibiotics, such as amoxicillin and clarithromycin/ metronidazole, and a proton pump inhibitor, such as omeprazole, gives a high eradication rate, and is now applied for clinical treatment of H. pyloriassociated gastroduodenal disease. However, eradication is not always successful and harmful side effects of these drugs may be encountered. Moreover, the acquisition by H. pylori of resistance to
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2. Experiments 2.1. Materials PVA (degree of saponification: over 98.0 mol; mean degree of polymerization: 1000, 1700 and 3210) (almost hydrolyzed PVA), paraffin oil, Span 80, hexane and ethyl acetate were purchased from Merck (India). Palmitoyl chloride, phosphatidyl ethanolamine, cholesterol and oleic acid were purchased from Sigma (USA). Acetohydroxamic acid was purchased from Fluka Chemicals (USA). The other chemicals were of danalytical gradeT and doubledistilled water was used for all experiments. 2.2. Preparation and characterization of PVA beads
s
Fig. 1. Recognition of lipobeads by PE specific surface receptors of H. pylori.
antibiotics, including clarithromycin and metronidazole, has also become a serious problem that may disturb treatment efficacy. Therefore, non-antibiotic agents, which are both highly effective and safe, are required to diminish H. pylori-induced gastric lesions. Ohta et al. [15] evaluated the effects of urease inhibitors acetohydroxamic acid on H. pyloriinduced gastritis in Mongolian gerbils. Bacterial infection rates were reduced to 40–50% of the control values of 100% by the highest dose of AHA. Since antibiotic-resistant strains of H. pylori have become serious problem, non-antibiotic, urease inhibitors may be very useful to control H. pyloriassociated gastroduodenal disease. AHA is a small molecule (molecular weight 75.07), it can permeate intact bacterial cells and effectively inhibits the urease activity of H. pylori.
PVA beads were prepared by emulsification followed by low temperature crystallization method described previously [14]. PVA powder was introduced in hot water having concentrations of 5%, 10%, 15% and 22% w/w. Then AHA was added in PVA solutions at room temperature and then emulsified in paraffin oil (containing 1.5% w/v surfactant, Span 80) to form beads. The beads were then cross-linked by slow freezing the suspension at 20 8C at various times (24, 48, 96 and 192 h) followed by a slow thaw at 4 8C for 2 days. After freeze thawing, the oily portion was removed by extraction with ethanol, hexane and ethyl acetate. After extraction, the hot air was bubbled through the PVA bare beads suspension and the residual amount of moisture was removed from the PVA bare beads. After drying, they were stored in dessicator. AHA is soluble in ethanol so after extraction with ethanol of PVA bare beads, the free AHA was removed. The particle size and size distribution of PVA beads were determined in a Zetasizer 3000 HS (Malvern Instrument, UK). Surface morphology of PVA beads was determined by scanning electron microscopy (SEM, Jeol JX 840-A, Tokyo, Japan). A thin film of aqueous dispersion of PVA beads was applied on double stick tape over an aluminium stub and air-dried to get uniform layer of particles. These particles were coated with gold to a thickness of about 450 2 using Sputter gold coater. The samples were scanned and photographs were taken (Fig. 2).
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Fig. 2. SEM microphotograph of PVA hydrogel beads.
Drug entrapment efficiency was determined by dissolving PVA bare beads in hot aqueous solution and the digested homogenate was centrifuged at 3000 rpm for 3 min. The solution was filtered and the filtrate was assayed for AHA colorimetrically at 503.5 nm in a Shimadzu 1601 UV/Visible Spectrophotometer. 2.3. Preparation and characterization of acylated PVA beads Surface acylation with fatty acid chains was accomplished by treating the PVA beads with 1 M palmitoyl chloride in hexane at room temperature for 2–3 days and the solvent was evaporated. The palmitic species became anchored on the beads via esterification with surface hydroxyls. The completion of this reaction was characterized using ATR-FITR. For analyzing the surfaces with these analytical tools, PVA films were formed by identical chemical treatment as described above.
7.4 was used as aqueous medium (total lipid concentration, 84 mM) to hydrate the dry lipid film. Then they were sonicated for 20 min resulting liposomes were then evaluated for average vesicle size. Mean particle size and polydispersity index of PE liposomes were determined in a Zetasizer (Malvern Instrument). Final formation of lipobeads was accomplished by combining equal parts of a suspension of surface modified beads with a PE liposomes suspension. Beads were added to the PE liposome dispersions with vortexing for 2 min in microfuge tubes. In this manner, the PE liposomes are reported to spontaneously collapse into a continuous bilayer surrounding the beads. After 10 min, the beads were then centrifuged and resuspended in buffer, repeating five times to remove unbound liposomes and free lipids, thus leaving single bilayer covered beads. Size distribution of lipobeads was measured in a Zetasizer (Malvern Instrument). To investigate the homogeneous distribution of liposomal bilayer on the PVA bare beads, confocal microscopy study was carried out. Rhodamine 123-PE liposomes were prepared by lipid cast film hydration method [16]. Rhodamine 123 bearing lipobeads were prepared by mixing equal parts of a suspension of surface modified beads with rhodamine 123-PE liposome suspension. Sample was viewed and imaged using a Leica LSM 510 system equipped with an argon ion laser. Additional excitation sources included HeNe1 and HeNe2 lasers for the respective 538 and 633 nm excitation wavelengths were performed at room temperature (~20 8C). Imaging experiments were typically performed using a 263/1.3 oil immersion objective with the diaphragms set to allow the probing of ~1 Am wide vertical slices of the imaged beads.
2.4. Preparation of lipobeads 2.5. Drug release studies PE liposomes were prepared by lipid cast film hydration method [16]; lipids, phosphatidyl ethanolamine (PE), oleic acid and cholesterol were dissolved in minimum quantity of chloroform in a round-bottom flask. A thin film of the lipids was cast on the inner surface of the round-bottom flask by evaporating the solvent under reduced pressure using rotary evaporator (Buchi type, York Sci., Mumbai). The flask was constantly rotated until dried film was formed. Final traces of solvent were removed under vacuum. 10 mM HEPES buffer containing 0.8% NaCl, adjusted to pH
The effect of PVA concentration, freezing time and PE bilayer on AHA release rate was observed. The drug release studies were carried out by USP dissolution apparatus paddle type at 37F1 8C. The dissolution fluid (400 ml) was simulated gastric fluid (SGF, pH 1.2). The PVA bare beads and lipobeads were held at the bottom of the vessel in a stainless-steel wire mesh (sinker). The paddle was positioned approximately 2.5 cm from the bottom of the vessel and was rotated at 50 rpm. An aliquot (2 ml) of
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dissolution fluid was removed at predetermined times and 2 ml of fresh fluid was added to the vessel to maintain the original volume. The concentrations of AHA were analyzed colorimetrically at 503.5 nm by using Shimadzu 1601 UV/Visible spectrophotometer. 2.6. Agglutination assay The bacterial strains H. pylori 1101 (isolated from a patient suffering from functional dyspepsia and shown to have erosive gastritis upon endoscopy) and H. pylori 69A (isolated from a patient with non-ulcer dyspepsia) were used in this study. H. pylori was grown on agar plates made of brain heart infusion agar containing 0.25% yeast extract, 10% sheep blood and supplemented with 0.4% Campylobacter selective complement (Skirrrow supplement, SR 69), maintained in a micro-aerophillic atmosphere (85% N2, 10% CO2, 5% O2) for 2 days, harvested under sterile conditions into brain heart infusion agar and finally diluted in Dulbecco’s modified Eagle’s medium plus Ham’s F12 (DMEM Ham’s 1:1) (pH 5.4) without antibiotics at 108 bacteria ml1. A bacterial cell suspension of H. pylori (2108, colony-forming units (CFU)/ml) in phosphate-buffered saline (PBS, pH 7.4) was prepared. This bacterial suspension was mixed with one-tenth of its volume of the lipobeads suspension, commercial PE and PVA bare bead suspension in a small glass test tube. The mixture was then incubated at 37 8C with or without shaking. 2.7. Adherence assay (radiolabelling assay) 14
C-labelled (1.7 ACi/ml) proline and histidine were added to the growth medium at the time of inoculation with bacteria. H. pylori strain 69A was used for all radiolabelling studies as described previously [17]. Incubation conditions were identical to those described above. Since 14C-labelled proline and histidine were used, gases which were released into the culture jar were absorbed in Protosol. After incubation for 12 h, bacteria were pelleted by centrifugation at 3000 rpm for 15 min, washed three times in sterile PBS (pH 7.4) and resuspended in PBS (pH 7.4) to an optical density of 0.5 at 600 nm. Incorporation of radioactive label into the bacteria was determined by counting disintegrations per min (dpm) in a h-scintillation counter.
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KATO-III cells obtained from the American Type Culture Collection (Rockville, MD) are gastric epithelial cells originally derived from a human gastric adenocarcinoma. The cells were grown in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum 20% v/v, penicillin 100 IU/ml and streptomycin 0.1 mg/ml. Cells were cultured in polystyrene tissue culture flasks in an atmosphere of 5% CO2 in air and 98% humidity at 37F1 8C. Confluent growth was obtained when flasks were incubated for 8–10 days, changing the growth medium at least once every 3 days. Cells were counted on a haemocytometer slide by phase-contrast microscopy; 104 cells/ml was initially attached to the polystyrene and 103 cells/ml remained in the suspension. KATO-III cells were subcultured and grown in 12well tissue-culture cluster dishes. After 8–10 days, the number of gastric epithelial cells adherent to polystyrene was approximately 3106 cells/well. H. pylori cells grown in supplemented Brucella broth for 14 h were resuspended in RPMI-1640 medium without antibiotics. The following suspensions were prepared, H. pylori incubated (1 h) with lipobeads (HP-LB), H. pylori incubated (1 h) with PVA bare beads (HP-PB) and H. pylori alone (HP). H. pylori suspensions were washed once in PBS (pH 7.4) and were added to KATO-III cells. For quantitation of H. pylori adherence to KATOIII cells, 108 3H-labelled bacteria were resuspended in 1 ml of RPMI-1640 medium, without antibiotics and added to the wells of a cluster dish. The cluster dishes were incubated in micro-aerophilic conditions for 3.5 h at 37F1 8C. Non-adherent bacteria were then removed by six washes with 1 ml of PBS (pH 7.4). To remove adherent H. pylori and KATO-III cells from the wells, trypsin 0.25% w/v was added and incubated at 37F1 8C for 10 min. The number of bacteria adherent to KATO-III cells and polystyrene surfaces was determined by counting dpm in 0.1 ml samples collected from each well after trypsination. Viability of KATOIII cells was determined at the beginning and end of the assays by trypan blue exclusion. 2.8. In situ adherence assay with human stomach cells Multiple samples of adult human oesophagus, stomach, duodenum and colon were obtained from the surgical pathology and autopsy files of the Dr.
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Periyar Government hospital, Madurai, India. Only samples of nondiseased tissues were used in these experiments. All tissues were fixed in 10% formalin or in a solution of picric acid/formaldehyde/glacial acetic acid (15:5:1, Bouin’s fluid) and subsequently embedded in paraffin [18]. Five micron sections were prepared and used for haematoxylin–eosin staining (to identify cell types present in gastric units and to verify that the tissue samples had no pathologic changes) and/or for subsequent adherence assays. Tissue sections were deparaffinized in xylene and isopropanol, rinsed in water followed by PBS (pH 7.4) and then incubated for 15–30 min in blocking buffer (0.2% bovine serum albumin/0.05% Tween 20, prepared in PBS (pH 7.4). The FITC-labeled bacterial suspension was diluted 20-fold in blocking buffers and 200 Al was placed on the slide, which was then incubated for 1 h at room temperature in a humidified chamber. Slides were subsequently washed four to six times with PBS (pH 7.4) prior to inspection. To analyze the ability of formulations to block binding, 200 Al suspensions of FITC-labeled bacteria were preincubated for 2 h at room temperature with lipobeads and PVA bare beads. Bacteria were washed once in blocking buffer before the mixture was added to tissue sections. After incubation, the in situ binding was observed by using fluorescence microscopy (Leica, Germany). 2.9. In vitro growth inhibition (GI) studies In vitro GI studies were preformed with H. pylori strains NCTC 11637. Percentage bacterial growth inhibition (%GI) was the parameter studied. H. pylori
was grown on agar plates made of brain heart infusion agar containing 0.25% yeast extract, 10% sheep blood and supplemented with 0.4% Campylobacter selective complement (Skirrrow supplement, SR 69), maintained in a micro-aerophillic atmosphere (85% N2, 10% CO2, 5% O2) for 2 days, harvested under sterile conditions into brain heart infusion agar and finally diluted in PBS (pH 7.4) without antibiotics at 108 bacteria ml1. The bacterial suspensions were centrifuged at 600 rpm for 30 min. These experiments were repeated thrice and the bacterial cell concentration approximately adjusted by dilution with PBS (pH 7.4) for measuring the absorbance at 660 nm. The following formulations bearing AHA (twice the minimum inhibitory concentration (MIC)—7 mM) were added to the H. pylori suspension to a final volume of 10 ml per sterile tube. 1. 2. 3. 4. 5.
AHA bearing PVA bare beads—PVA bare beads AHA bearing lipobeads—Lipobeads Drug free PVA bare beads—Placebo I Drug free lipobeads—Placebo II Plain AHA—AHA
The tubes were incubated with constant agitation at 37F1 8C under micro-aerophilic conditions. In order to check any antimicrobial activity of constitutive ingredients, the drug free PVA bare beads (Placebo I) and lipobeads (Placebo II) were taken for the studies. The bacterial growth was monitored spectrophotometrically at 660 nm in a Shimadzu 1601 UV/Visible spectrophotometer against blank (uninoculated broth) at 6, 12 and 24 h. This procedure was repeated thrice.
3. Results and discussion Lipobeads may potentially be useful in several novel biomedical applications. As a drug carrier, lipobeads should be superior to liposomes in the sense of enhanced mechanical stability. Long-term storage is possible since acylated beads loaded with drug can be stored in a dry form before lipid coating. Also, many of the techniques developed for liposomes such as stimuli-sensitive drug release, steric stabilization and receptor- or antibodymediated drug targeting [19] should still be applicable with lipobeads. As with other human pathogens, it has been proposed that H. pylori species express multiple adhesins that would allow bacterial interaction with various target cell epitopes on mucin components including fucosylated glycoconjugates and phosphatidyl ethanolamine [11]. For effective H. pylori eradication, the drug delivery systems must plug and seal the H. pylori as well as continuously release the antibiotics at the infection site in effective concentration and in fully active form.
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Development of PE liposomes in H. pylori treatment has many challenges. PE constitutes one such class of lipids, which when dispersed in pure form under physiological conditions assemble into nonbilayer structures. This is in contrast to lamellar phase forming lipids, which readily form hydrated lamellar phase or bilayer structures when dispersed in aqueous media [20]. We developed liposomes anchored PVA hydrogel beads to overcome the limitations of the PE liposomes in H. pylori treatment as described previously [14]. One approach has been to create a hydrophobic anchor for the bilayer by attaching lipid molecules to the surface of preformed PVA xerogel (dry hydrogel) beads. When the surface modified xerogel is hydrated and treated with a PE liposomes suspension, the hydrophobic lipid molecules and other intrinsic membrane components of the PE liposomes associate spontaneously with the hydrophobic fatty acid anchors on the surface of the polymer and self-organize into a membrane distributed over that surface through hydrophobic interactions. 3.1. Preparation and characterization of PVA beads PVA beads were prepared by emulsification followed by low temperature crystallization method and this procedure produced PVA xerogel spheres ranging from 50 to 100 Am Maximum size distribution of lipobead was 75F10 Am. Spherical beads ranging in size from 80 to 100 Am were selected by sieving for further modification. Lipobead yield after sieving was 68.3%. The surface morphology of PVA hydrogel beads was investigated by scanning electron microscopy that revealed the spherical shape with smooth surface (Fig. 2). The drug entrapment efficiency was increased with PVA concentrations. At 22% w/w PVA concentration, the entrapment efficiency (65.3%) was high when compared to 5%, 10% and 15% w/w concentration. 3.2. Preparation and characterization of acylated PVA beads Surface acylation with fatty acid chain was accomplished by treating PVA bare beads with 1 M palmitoyl chloride in hexane at room temperature for 2–3 days. The completion of reaction was characterized by using attenuated total reflectance ATR-FTIR spectroscopy study. In brief, the infrared beam enters the ATR crystal from one of the side faces. If the refractive index of the crystal is higher than the PVA, and the incident angle of the beam is higher than a critical angle, then the infrared beam is totally reflected at the crystal/PVA interface, and the beam travels inside the crystal and exits from the other side face. However, due to diffraction at the crystal surface, a small fraction of the beam penetrates into the PVA layer and is absorbed by PVA. The fraction of the beam which is absorbed gives rise to absorption bands in the ATR spectrum and is used to monitor the concentration of each component within the penetration depth in the polymer cage. In ATR-FTIR spectrum of palmitoyl chloride anchored PVA, the band at 1776 cm1 was for ester group which was formed between the carboxyl group of palmitoyl chloride and the hydroxyl group of PVA (data not shown). The formation of an ester bond was determined and 53% changes in atomic ratio at the surface were measured by ATR-FTIR. 3.3. Preparation and characterization of lipobeads PE liposomes prepared were studied by light microscopy and transmission electron microscopy. The electron microphotographs showed that liposomes were unilamellar vesicles and composed of bilayer unit membranes, about 75 2 thick. Mean diameter of PE liposome formulation was 4.2F1.2 Am. Final formation of lipobeads was accomplished by combining PVA bead suspension to a PE liposome suspension. Anchoring of PE liposomes on PVA hydrogel beads was confirmed by swelling studies. When dried acylated beads are placed in water, their diameter increases about one to four times at swelling equilibrium. However, the beads aggregate and float on the water surface due to their surface hydrophobicity. Adding a liposome suspension to the solution with floating beads causes the beads to separate and sink to the bottom.
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We measured contact angle of PVA bare beads, acylated PVA beads and lipobeads. For acylated PVA surfaces, the contact angle was 1058 indicating that the surface is hydrophobic. The contact angle of the surface was reduced to less than 158 after the film was treated with liposomes. When beads are separated from water by filtration and exposed to air, the bilayer is disturbed and the beads again became hydrophobic. The contact angle of the beads again became N1008. Fig. 3 shows a confocal fluorescence microphotograph of a homogeneous lipid bilayer surrounding a lipobead when viewed as a 1 Am slice at the focal depth at the equator of a bead. From this photograph, we concluded the homogeneous distribution of bilayer on the PVA bare beads. An advantage of using beads as a platform for PE bilayers supported assemblies is the direct exposure of the number of PE sites that are readily afforded by H. pylori receptors. In these experiments, varying the mole fraction of PE that is mixed with oleic acid and cholesterol regulates the number of PE molecules per bead. Thus bead supported bilayers derived from a mixture of PE, oleic acid and cholesterol of known stoichiometry can result in a bead population with a reasonably defined surface coverage of PE. This is established by dividing the surface area of the bead (4pr 2) by the area of the average lipid head group. The desired surface coverage of PE is then simply affected by using appropriate mole fraction of PE in the vesicle preparation. By extension of this approach, one can also ensure that an excess amount of lipids is always mixed with beads. 3.4. Drug release studies In vitro release studies of AHA were carried out with PVA bare beads which were prepared at various freezing times and PVA concentrations. Fig. 4A shows the release profiles of AHA from PVA bare beads (22% w/w PVA, pH 1.2), which were prepared at various freezing times at 20 8C. The release rate of AHA was slower on longer freezing times. Fig. 4B shows the effect of PVA concentrations at pH 1.2 on the release profiles of AHA. Slower release rate of AHA was observed at 22% w/w concentrations of PVA. To investigate the effect of PE bilayer on in vitro drug release rate of AHA, both lipobeads and PVA bare beads were taken. Fig. 4C shows the effect of bilayer on the release profile of AHA from PVA bare beads. AHA was released at higher rate from PVA bare beads than lipobeads. From Fig. 4C, it was concluded that PE bilayer significantly slower/control the AHA release rate. Till 6 h, the release rate was significantly lower with lipobeads when compared to PVA bare beads. After 6 h, difference between the release rate of PVA beads and lipobeads was decreased. Due to the hydrophilicity of the PVA bare beads,
Fig. 3. Confocal fluorescence photomicrograph of lipobeads.
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Fig. 4. Release profiles of AHA from PVA bare beads at various (A) freezing times (PVA concentration 22% w/w) and (B) concentrations of PVA (freezing time 48 h). (C) Effect of PE bilayer on release rate of AHA from PVA bare beads (PVA concentration 22% w/w).
the drug release rate was higher whereas due to the hydrophobicity of lipobead formulations, the drug release rate was slower up to 6 h. Similar release rate of PVA bare beads and lipobeads might be due to the reduction in hydrophobicity of lipobeads after 6 h. Based on in vitro drug release studies and drug entrapment efficiency, PVA bare beads prepared at 22% w/w PVA concentration (freezing time 48 h) were taken for further studies.
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3.5. Agglutination assay H. pylori showed different agglutination reactions with various formulations. Among the three formulations tested—lipobeads, PVA bare beads and commercial PE—strong agglutinations were observed with the specific lipobeads and commercial PE (Table 1). All H. pylori organisms were agglutinated by the lipobeads whereas PVA bare beads were found to be non-reactive with H. pylori strains. The agglutinating ability of lipobeads towards H. pylori might be due to the retention of PE specific receptors on H. pylori surface. It is interesting to note that PE on the lipobeads as well as commercial PE was recognized by all H. pylori strains. Lipobeads showed the same binding characteristics with PE specific surface receptors of H. pylori as the commercial PE. It revealed the intact specificity of homogeneous distribution of PE liposomes on the PVA bare beads. It further confirmed the previous report that PE is more likely to be function as a receptor for H. pylori related adhesion [11]. 3.6. Adherence assay (radiolabelling assay) In the present study, we have demonstrated the attachment of H. pylori to epithelial cells of gastric origin in vitro. Adherence of H. pylori to KATO-III cells was morphologically identical to the in vivo observations reported previously [17]. In the present bacterial adherence assays, the number of attached labeled bacteria was determined by measurement of radioactivity. As H. pylori requires incubation for z5 days to achieve adequate growth on agar media, a rapid and reproducible method for quantitation of bacterial adherence with radiolabelled organisms was employed in this study as described previously [17]. Minimal binding of radiolabelled bacteria to plastic alone and the negligible loss of radioisotope from H. pylori both indicated that free radioisotope was not an important confounding variable in these experiments. Bound radioactivity of HP and HP-PB with KATO-III cells was greater than HP-LB and the number of H. pylori adhering to KATO-III cells was similar with HP and HP-PB (Fig. 5). Adherence of H. pylori to tissue culture cells was significantly reduced with HP-LB, because PE receptors of organisms were completely plugged and sealed by the HP-LB formulations. The reduction in bacterial binding to KATO-III cells with HP-LB was probably related to less availability of PE receptor as previously observed by Bitzan et al. [21]. They showed that bovine colostrum completely blocked the bacterial attachment to PE, Gg4 and Gg3. Colostral lipid extracts of bovine colostrum contained PE and lyso-PE that bound H. pylori in vitro. Similarly, lipobeads completely plug and seal the PE receptor and block the H. pylori adhesion with KATO-III cells. 3.7. In situ adherence assay with human stomach cells In situ adherence assay should be useful in further characterizing the H. pylori adhesin receptors and for identifying therapeutically useful compounds that inhibit strain specific and cell-lineage specific binding of this human pathogen. Falk et al. [22] developed this experiment to characterize the surface receptors present on H. pylori. We followed the same reported and validated protocol for in situ adherence assay to evaluate the targeting propensity of lipobead formulation. The drug-free formulations, i.e. lipobeads and PVA bare beads, were incubated with H. pylori strains for 2 h. The following three samples were overlaid on sections of Table 1 Lipobeads agglutination patterns of H. pylori (n=3) Formulations
H. pylori strain 69A
H. pylori strain 1101
Lipobeads PVA bare beads Commercial PE
+++ +++
+++ +++
The agglutination reaction was scored as follows: +++ = strong positive reaction with large clumps; ++ = strong-positive reaction with moderate size clumps; + = positive agglutination with fine clumps; w = weak agglutination; = no agglutination.
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Fig. 5. Radioactivity of adherent HP-LB, HP-PB and HP with KATO-III cells. HP-LB- H. pylori suspension preincubated with lipobeads. HPPB- H. pylori suspension preincubated with PVA bare beads. HP – H. pylori suspension.
human stomach cells: H. pylori incubated with lipobeads (HP-LB), H. pylori alone (HP) and H. pylori incubated with PVA bare beads (HP-PB). The fluorescence microphotographs reveal the binding intensity of H. pylori samples with human gastric mucosa. Fig. 6A indicates that HP was strongly bound at surface mucous cells situated in the human stomach portion. The intensity of binding was significantly higher in HP compared to that of HP-LB (Fig. 6A and C). HPPB showed equal intensity of binding like H. pylori with the human stomach cells (Fig. 6B). The intensity of binding was almost nil in HP-LB (Fig. 6C). The results clearly revealed that the PE receptors were completely bound with the lipobeads. Therefore HP-LB strain could not bind with the human stomach cells. PE specific receptors are intact in the case of HP-PB and HP suspensions, thus they strongly bind with the human stomach cells. In situ adherence assay confirmed the superior binding propensity of lipobeads with PE specific surface receptors of H. pylori. 3.8. In vitro growth inhibition studies In the present study, lipobeads–H. pylori interaction and adsorption to the bacterial cell surface could be exploited for effective localization and targeting of lipobeads to a preselected bacterial cell line. The method selected for the proposed study was turbidometry based on optical density measurements [18]. Quantitation of lipobeads– bacterial interaction was approached in terms of percentage growth inhibition (%GI). One of the major advantages of culture is that it allows sensitivity testing of H. pylori to the antimicrobial agents used in the treatment. The effect of different drug-loaded and drug-free formulations on bacterial growth was investigated. It was reported that AHA specifically inhibits the urease enzyme, which is secreted by the H. pylori [23]. The antimicrobial effect of formulations was determined in terms of percentage growth inhibition (%GI) that was calculated as the ratio of optimal density (OD) (at 660 nm) of a given mixture against that of tubes containing H. pylori alone and can be expressed as given below: %GI ¼
OD of test organism at a particular time interval OD of test mixture at the same time interval 100 OD of test organism at a particular time interval
All of these results of these experiments are present in Fig. 7. The results could be arranged to an order of %GI performances: Lipobeads N AHA N PVA bare beads N PlaceboI N PlaceboII
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Fig. 6. Section of human adult stomach incubated with FITC-labeled (A) HP (H. pylori alone), (B) HP-PB (H. pylori incubated with lipobeads) and (C) HP-LB (H. pylori incubated with PVA bare beads) (450).
Placebo I (drug-free PVA bare beads) and Placebo II (drug-free lipobeads) exhibited insignificant growth inhibition (Fig. 7), which suggested that constitutive ingredients have no antimicrobial activity. They can only interact with the bacterial cell surfaces like cell adhesion of PE and interaction of PVA hydrogel conjugates on the tight junction of epithelium cells. Both PVA bare beads and lipobeads have the drug concentration equivalent to MIC (3.5 mM). After 6 h, %GI values were 35F4%, 25F4% and 100% for AHA, PVA bare beads and lipobeads, respectively. The bacterial growth was inhibited remarkably by lipobeads than PVA bare beads and drug (AHA). Lipobeads showed superior %GI than the equal amount of drug bearing PVA bare beads and AHA (Fig. 7). It could be assumed that the affinity of lipobeads towards phosphatidyl ethanolamine specific receptors present on H. pylori surface might be instrumental in pursuing better results.
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Fig. 7. Percentage growth inhibition of formulations.
The %GI values of formulations at different time intervals are shown in Fig. 7. The PVA bare beads completely inhibit the bacterial growth only after 24 h. Although the drug release rate of lipobeads was slower than PVA bare beads (Fig. 4C), lipobeads were completely inhibiting the H. pylori growth within 6 h. Thus, the %GI of lipobeads was independent of drug concentration and incubation time. This could be due to the fact that drug targeting was predominate as a result of the specificity of the lipobeads towards the PE specific receptors on the bacterial surface glycocalyx. PE–H. pylori interaction could lead to increase in drug concentration as well as therapeutic index at the H. pylori surface.
4. Conclusions
Acknowledgements
The characterization studies suggested that lipobeads could be a novel delivery device to improve the bioavailability of anti-H. pylori agents which are aimed to produce a local and specific effect in the stomach and are specifically absorbed through the upper region of the stomach. The plugging and sealing effect of the lipobeads could also impart superior targetability on the H. pylori surface. The proposed site specific drug delivery system targets the H. pylori more effectively and could serve to optimize antibiotic monotherapy of H. pylori based infections. These systems could protect the drug from the gastric environments. From all of the experiments performed, it can be suggested that the developed lipobeads could be successful in the treatment of H. pylori. In vivo growth inhibition studies of drug bearing lipobeads with animal models in order to evaluate the efficiency of formulations in vivo may further substantiate the present conclusions.
Authors would like to acknowledge Dr. Yegneswaran, Deputy Director, Regional Research Laboratory, Bhopal, India and Director, Madurai Kamarajar University, Madurai, India for providing necessary facilities. R.B. Umamaheshwari acknowledges Indian Council of Medical Research, New Delhi, India for awarding Senior Research Fellowship during the tenure of this work.
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