Review

. . . .

. . . .f H. pyltations.to erad. . . .. . . .. . . .. . . .

3.2.3. Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

. . . . . . . . . 183

. . . . . . . . . 183

Journal of Controlled Release 189 (2014) 169186

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre lAbbreviations: AA, acrylic acid; AGS cells, human gastric adenocarcinoma cell line; AHA, acetohydroxamic acid; AS OND, antisense oligonucleotides; AuChi, chitosan-modied gold3.4.3. Bismuth compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.4.4. Iron microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.4. Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1773.3. Hybrid systems with liposomes and polymeric particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

3.3.1. Polymeric core coated with a phospholipid bilayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1823.3.2. Liposome coated with biocompatible polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

3.4. Metallic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1833.4.1. Zinc nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1833.4.2. Silver nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183nanoparticles; BPO, benzoyl peroxide; Ch, cholesterol; Co170; GME, Garciniamangostana extract; GNP, gliadin nanoacid; MPs, microparticles; NIPASM, N-isopropylacrylamidTEGDMA, triethyleneglycol dimethacrylate; UEA 1, Ulex E Corresponding author at: Rua de Jorge Viterbo Ferreir

E-mail address: shreis@ff.up.pt (S. Reis).

http://dx.doi.org/10.1016/j.jconrel.2014.06.0200168-3659/ 2014 Elsevier B.V. All rights reserved.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

3.2.1. Polyacrylic acid . . . . .3.2.2. Proteins . . . . . . . .Contents

1. Introduction . . . . . . . . . .2. Treatment of H. pylori infection .

2.1. Overview of the discovery o2.2. Current therapy and its limi

3. Micro- and nanotechnology applied3.1. Liposomes. . . . . . . .

3.1.1. Simple liposomes3.1.2. Double liposomes

3.2. Polymeric particles . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170ori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171ication of H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174Drug deliveryEradication of Helicobacter pylori: Past, present and future

Daniela Lopes a, Cludia Nunes a, M. Cristina L. Martins b,c, Bruno Sarmento b,d, Salette Reis a,a REQUIMTE, Departamento de Cincias Qumicas, Faculdade de Farmcia, Universidade do Porto, Porto, Portugalb INEB Instituto de Engenharia Biomdica, Universidade do Porto, Porto, Portugalc ICBAS Instituto de Cincias Biomdicas Abel Salazar, Universidade do Porto, Porto, Portugald IINFACTS Instituto de Investigao e Formao Avanada em Cincias e Tecnologias da Sade, Instituto Superior de Cincias da Sade-Norte, Gandra-PRD, Portugal

a b s t r a c ta r t i c l e i n f o

Article history:Received 3 April 2014Accepted 13 June 2014Available online 23 June 2014

Keywords:Helicobacter pyloriTreatmentNanoparticlesMicroparticles

Helicobacter pylori is the major cause of chronic gastritis and peptic ulcers. Since the classication as a group 1carcinogenic by International Agency for Research on Cancer, the importance of the completeH. pylori eradicationhas obtained a novel meaning. Hence, several studies have beenmade in order to deepen the knowledge in ther-apy strategies. However, the current therapy presents unsatisfactory eradication rates due to the lack of thera-peutic compliance, antibiotic resistance, the degradation of antibiotics at gastric pH and their insufcientresidence time in the stomach. Novel approaches have been made in order to overcome these limitations. Thepurpose of this review is to provide an overview about the current therapy and its limitations, while highlightingthe possibility of using micro- and nanotechnology to develop gastric drug delivery systems, overcoming thesedifculties in the future.

2014 Elsevier B.V. All rights reserved.n A, conconavalin A lectin; CTB, cholera toxin B subunit; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocoline; E170, epikuronparticles;H. pylori,Helicobacter pylori; HEM, hydroxyethyl methacrylate; HPMC, hydroxy propylmethyl cellulose; LLA, linolenice; NPs, nanoparticles; PE, phosphatidylethanolamine; RBC, ranitidine bismuth citrate; SA, stearylamine; SPs, small particles;uropaeus agglutinin I lectin; -PGA, poly--glutamic acid.a, 228, 4050-313 Porto, Portugal. Tel.: +351 220428672; fax: +351 226093483.

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

1. Introduction

Helicobacter pylori (H. pylori) is a gram-negative bacterium, usuallyin a spiral-shaped form, that can be converted in coccoid cells under ahostile environment [1,2]. These bacteria present several structuraland morphological characteristics that favor their penetration withinthe mucosa and consequently the colonization of the gastric antrumand the human duodenal mucosa [2,3]. Their major virulence factorslie on four to six agella enhancers of their mobility, urease production,phospholipase secretion, cytotoxin production and their ability to ad-here to the target cells [13]. These virulence factors enable theirmobil-ity through the gastric mucus and the colonization of the surfacebetween the mucus gel layer and the epithelial cells [3,4]. Adhesinsare responsible for the adherence to carbohydrates of the mucosa andto epithelial cells, namely through the adhesion to polysaccharides,laminins and Lewis b antigen among others, playing an important rolein the pathogenesis of the bacteria [36].

Although without a clear explanation, other extradigestive conditions,namely idiopathic thrombocytopenic purpura, iron deciency anemia,ischemic heart disease, stroke, Parkinson's disease and Alzheimer's dis-ease have been recently related to the presence of H. pylori [11].

The importance of the therapy in clinical manifestation ofH. pylori isunquestionable. However, despite all the endeavors, the current thera-py presents many limitations which have led to the failure of H. pylorieradication. This reviewprovides an overview about the traversed path-way until the current therapy and its limitation. Furthermore, a summa-ry of all the reports with micro- and nanoparticles applied to gastricdelivery through active or passive targeting to the bacteria or throughmucoadhesiveness to the gastric mucosa will also be discussed.

2. Treatment of H. pylori infection

2.1. Overview of the discovery of H. pylori

170 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186Currently, the worldwide population infected is around 50%, beingeven higher in developing countries [7]. Prevalence rates of H. pylori in-fection varies according to race/ethnicity, socioeconomic conditions andage, being highest with aging [7]. Commonly, their colonization isasymptomatic, resulting only in histological signs of chronic gastritis[8,9]. However, approximately 20% of the infected population evolvesinto a clinical condition, commonly chronic gastritis and peptic ulcer[8,9]. This incomprehensive and complex H. pylorihuman relationship,where only a portion of the infected peoplemanifests a disease, have ledto a controversy about the seriousness of H. pylori infection [8]. Never-theless, the risk resulting from an unsuccessful eradication is higher inthe cases of clinical manifestation, since persistent infections may leadto gastric cancer, such as gastric mucosa-associated lymphoma tissueand adenocarcinomas [7,8,10]. In fact, bacteria eradication in patientswith low-grade lymphomas often results in the remission of the cancer[7]. According to these facts, the International Agency for Research onCancer (IARC), subordinated to the World Health Organization(WHO), declared H. pylori as a human carcinogenic (group 1) [7].Fig. 1. Timeline of the H. pylori discovery and the pThe discovery of H. pylori resulted from a slow and gradual progres-sion (Fig. 1). The rst report about gastric ulceration was written in1586 by an Italian physician named Marcello Donati [1,12,13]. Duringseveral years, the pathogenesis of this disease was exclusively associat-ed to stress and dietary factors, being treated by resorting to bed restand special diet [1]. In the second half of 18th century, the use of bis-muth compounds, namely bismuth subnitrate, to treat gastric ulcers be-came very popular as a result of the work of Gorham and Kussmaul [13,14]. Actually, bismuth compounds have antibacterial properties thatwere unknown at that time [13,14].

In 1875, Bottcher and Letulle noticed the existence of bacteria inulcer margins and suggested their relation to gastric disease [14]. How-ever, the presence of spiral organisms in human gastric washings wasreported by W. Jaworski, a Poland professor, only in 1889 [15]. He alsotheorized that the bacteriamay be related to the development of gastriculcers [1]. Nevertheless, his researchworkwas poorly publicized since itwas written in Polish [15]. The rst recognized report appeared only inthe latter half of the 19th century, when Bizzozero observed therogress of the therapy against the bacterium.

reaching the surface between the mucus gel layer and the epithelial

171D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186presence of spirochetes in canine gastric mucosa [2]. Bizzozero hy-pothesized that the bacterium could turn off acid secretion or toleratethe acidic environment [16]. Bizzozero's work was followed by Salo-mon, through the discovery of the propagation of spiral bacteria fromdogs and cats to mice in 1896 [13,15]. These ndings are the ground-work of the current studies of vaccines using H. felis-infected mouse[2]. Afterwards, spirochetes were found in the stomach of patientswith gastric carcinoma by W. Krienitz, in 1906 [13].

In 1910, the Croatian Karl Schwarz pronounced the famous phraseno acid, no ulcer that lead to the use of antacids (e.g. magnesiumand aluminium hydroxide) for symptomatic relief of ulcers in 1915[12,14]. In 1919, Kasai andKobayashi reported the presence of the spiralbacteria in several mammals, recognizing that it could cause hemor-rhagic erosionswhich could be healed by resorting to several antimicro-bials [16]. One year later, Osler andMcCrae described an epidemic acuteinfection in children, characterized by a vomitingwith neutral pH of thegastric juice, denominated hypochlorhydria [2]. In 1924, Murray Luckstudied the urease enzyme found in gastric mucosa, mistakenly believ-ing that it was naturally produced by gastric mucosa cells [17]. In the1950s, the study of urease continues with the research work of Fitzger-ald andMurphy, who found gastric urease in patients with peptic ulcersand believed that urease was produced to protect gastric mucosa [17].

Although participants in a society that considered the stomach as asterile environment due to the acidic environment, Allende andLykoudis reported the treatment of peptic ulcers with penicillin(1951) and streptomycin (1958), respectively [1]. However, this hy-pothesis was rejected by the medical community [1]. During the yearof 1957, Charles Lieber and LeFevre discovered that the treatmentwith antibiotic promoted the decrease of the conversion from urea toammonium [16]. They concluded that the gastric ureasemust be relatedto the presence of the bacteria [16]. Around 1966, H2-antagonists cameto be used to improve themanagement of gastric symptoms as a conse-quence of the discovery of gastric histamine receptors [12]. During the70s, several Spanish and Chinese physicians reported efcacy of furazol-idone and metronidazole to treat patients with ulcers [16].

The interest in these spirochetes began to spreadworldwide, involv-ing researchers from several countries, who reported the presence ofthe bacteria in the human gastric mucosa and noticed the curing of gas-tric diseases using antibiotic therapy [16,18]. However, in the 1960s and1970s, physicians and microbiologists believed that the stomach wassterile since they obtained negative bacterial cultures and therefore allthe evidences of the presence of spirochetes in the stomach wereundervalued [17].

Against the skepticism of almost all the scientic community, RobinWarren and Barry Marshall believed that there was a direct associationbetween the bacteria and gastric ulceration [13]. Evidences that servedas clues included the presence of spiral bacteria in the stomach and itspossible relation to gastric urease, epidemic hypochlorhydria and the ef-cacy of antibiotics to treat peptic ulcers [16]. Warren andMarshall no-ticed the immune response in hosts of H. pylori and describedmicrobiological properties of these bacteria, including the similaritywith the Campylobacter species [14]. During Easter break, a plate was in-cubated during 5 days,more than the usual attempt of 3 days, andwhenMarshall returned to the laboratory he foundnumerous colonies of Cam-pylobacter-like organism [15,17]. Firstly named Campylobacter pyloridisand afterwards corrected to Campylobacter pylori, the bacterium is cur-rently named Helicobacter pylori as it is a completely different genus[1,15]. In 1983, they reported in Lancet the rst culture of the bacteriaand, in 1985, Marshall ingested cultures of H. pylori in an attempt to ful-ll the Koch postulate, promoting gastric symptoms healed afterwardsby resorting to antibiotics and bismuth salts [1,15,19]. The magnitudeof this discovery is a direct consequence of the persistence against theacid-induced ulceration dogma and skepticism, being recognized in2005, by the award of the Nobel Prize in Physiology orMedicine [13,18].

In 1986, an initiatory review of omeprazole was published, opening

the use of the proton pump inhibitor drugs [18]. The treatment ofcells, where the H. pylori resides [3,4]. Although drug solutions reachthe gastric luminal region, their absorption into deeper layers of the gas-tric mucosa is hampered by the mucous layer barrier [28].

In order to increase the efcacy of H. pylori eradication, different pro-posals have been made, namely a bismuth-containing quadruple (BCQ)therapy, sequential and concomitant treatment and the use of novel an-tibiotics, such as rifabutin [11,29]. However, these options may have totake into account that the complexity of the treatment plan, includingthe switch halfway in the sequential treatment and the large numberof pills in concomitant and BCQ therapy, may decrease therapeutic com-pliance [11,29]. To overcome these limitations, novel effectivetherapies have been proposed: probiotics [30,31], phytomedicine [31],gastroretentive systems, namely oating drug delivery systems [32,33]and in a preventive approach, the attempt to develop an effective vaccine[23]. One of the foremost promising therapies that have recently emergedis based on the use of micro- or nanoparticles for direct contact with theH. pylori, through drug delivery techniques or mucoadhesive properties.H. pylori infection was improving from a double ineffective therapy,combining a PPI plus clarithromycin or amoxicillin to the current tripletherapy recommended by guidelines in Europe andNorth America sincethemid-1990s [1,20]. However, the rst report of the resistance tomet-ronidazole had already been published in 1985, followed by reportsmentioning the resistance to other antibiotics, namely -lactams, tetra-cyclines, uoroquinolones, rifamycins and nitrofurans [2,21].

In 1994, the US National Institute of Health recognized H. pylori asthemain cause of peptic ulcers and, in the same year, it was categorizedby the World Health Organization as a carcinogenic (group 1) [1,7].With the recognition of the foremost role ofH. pylori in the origin of gas-tric diseases, thousands of research works have been published aboutthe microbiology of the bacterium, including the sequence of the ge-nome (1997), novel virulent factors and mechanisms of resistance toantibiotics [1]. Improvements in diagnostic tests and prophylacticmethods (vaccines) are also being studied [1]. Nowadays, one of theforemost research subjects is the improvement of the current therapy.

2.2. Current therapy and its limitations

The treatment plan currently adopted as arst-line option includes acombination of a proton pump inhibitor, clarithromycin and amoxicillinor metronidazole/tinidazole, according to International Guidelines [10,22]. This therapy persists during 7 to 14 days, twice a day [10]. Eradica-tion rates of H. pylori treated with a 14-day triple therapy reached only70% in non-ulcer dyspepsia patients and 80% in patients with pepticulcer [10]. In Europe, Asia and North America rates of 20 to 45% havebeen reported [23]. This eradication rate is distant from the desirablerate to infectious diseases and from that proposed by the WHO [22,24]. The main limitation of the current therapy results from the lack oftherapeutic compliance, due to the incidence of side effects and the dis-comfort resulting from the multiple doses [25,26]. This factor may leadto the development of antibiotic resistance [26]. Moreover, antimicrobi-al agents such as amoxicillin and clarithromycin are degraded by gastricacid [24]. Therefore it is necessary to use higher doses,which is reectedin the increase of gastrointestinal side effects, namely diarrhea, nausea,vomiting, bloating and abdominal pain, and consequently thediscontin-uation of the therapy [26]. Another important reason is the antibiotic re-sistance thatH. pylori has been developing, for instance the resistance tometronidazole has reached around 40% in developed countries and ex-ceeds 90% in developing countries [27]. The resistance to clarithromycinhas also been increasing, reaching more than 20% in southern Europe[23]. The bacteria are sensitive to other antimicrobial drugs, neverthe-less they cannot be used in acidic medium [24]. Notwithstanding, theantibiotic residence time in the stomach is insufcient to achieve signif-icant concentrations capable of crossing the gastric mucosa andThis review will summarize all of the assays reported using micro/

nanoparticles applied to gastric delivery in order to increase H. pylorieradication rates.

3. Micro- and nanotechnology applied to eradication of H. pylori

Small particles (SPs), more specically microparticles (MPs) andnanoparticles (NPs), have unique physical and chemical propertiesresulting from their small size, such as the high surface-to-volumeratio and their reactivity [34]. According to the concept used by thema-jority of the authors cited, the terms microparticle and nanoparticlewill be used to refer to particles with a diameter of 1999 m and 1999 nm, respectively. Each of these particles can be manipulated inorder to achieve size, shape, chemical characteristics and specic li-gands enhancers of molecular interactions [34]. For instance, positivelycharged particles may be attracted to gastric mucosa, since it is nega-tively charged due to several surface groups, viz. sialic acid, carboxylor sulfate groups [28,35]. Additionally, H. pylori also is negativelycharged, which may play an important role in the interaction with SPs[24]. Faced with the serious emerging problem of bacterial resistanceto antibiotics, several antibiotic-loaded SPs have proven their usefulnessand efcacy both in vitro and in animal models [36]. SPs also allow asustained therapy since they can achieve higher retention time in thehuman body compared with small molecules of antibiotics [37]. Ap-proaching the ideal magic bullet, it is possible to use this technologyto target almost exclusively the bacteria, allowing the use of higherdoses without increasing side effects [38]. Some authors defend thatsize plays a central role in the SPs' diffusion into the gastric mucosa toreach H. pylori, since NPs with more than 200 nm have a decreased dif-

and present lower toxicity and interaction with the immune system[42]. Polymeric microparticles have also been used as drug carriersdue to the possibility to use mucoadhesiveness to target mucus and in-crease the retention time in the stomach [43]. It is also defended that SPscannot be excessively small to avoid internalization by gastric cells andto enhance theH. pylori:particle ratio [44]. The relation betweenMP andNP size and the length of a H. pylori bacterium are demonstrated inFig. 2.

Other advantages of encapsulated antibiotics include the controlla-ble release and identical distribution in the infected tissue, the increaseof pharmacokinetic properties of antibiotics, namely lipophilicity, andthe reduction of collateral effects leading to the increase of therapeuticcompliance [37]. Another pharmacokinetic limitation that may be over-come with drug delivery is the degradation of antimicrobial drugs byacidic pH. In fact, the pH of the stomach lumen is about 1 to 2 [45]. How-ever, adding the secretion of HCO3 molecules to the capability of themucus layer to resist to protons diffusion, a pH gradient is establishedfrom acid in the lumen to near neutral at the interface between themu-cosa and epithelial cells (Fig. 2) [45,46]. Hence, the pH of the gastricmucus layer varies between 4 and 6.5, with the exception of occasionalacid decreases to pH b 2when themucus layer is injured [47]. Addition-ally, the production of urease by the bacterium results in the productionof ammonia, maintaining periplasmic and cytoplasmic pH of the bacte-rium near to neutral even in the presence of acid shocks [1,47]. There-fore, the release of the antimicrobial drug near the site of infection ofH. pylori may protect the drug from acid degradation. Furthermore, itis improbable that bacteria might develop resistance to SPs because nu-merous and complex gene mutations would be necessary to overcome

172 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186fusion [24,39,40]. In fact, Hasani et al. (2009) referred to a size-dependent particle deposition in the inamed tissue of gastric ulcers,observing that NPs of 50 nm present higher adhesion than NPs of750 nm [39]. Additionally, some authors believe that submicrometersize is the key to the bacteriostatic effect of nanoparticles since theirability to penetrate the damaged bacterial membrane depends ontheir size [41]. On the other hand, NPs have an evident tendency to pre-mature drug leakage [39]. Additionally, it is argued that microparticles,such as magnetic particles, are also able to pass between epithelial cellsFig. 2. Use of micro- and nanotechnology to active and passive targeting of H. pylori, highlightieither the multiple mechanisms of action of antimicrobial SPs as wellas the possibility to combine different antimicrobial drugs in the samecarrier [38]. Their micro- and nanosize can also be used to develop vac-cines since they are recognized by the immune system through theirsimilarity to the bacteria and virus size [37]. Hence different particles(MPs and NPs) have been studied in order to increase the eradicationrate of H. pylori infection. These novel systems will be the focus of thisreview, particularly liposomes and polymeric, magnetic and metallicsystems.ng the relation between the size of MPs and NPs and the length of the H. pylori bacterium.

3.1. Liposomes

Liposomes are spherical vesicles composed of amphiphilic lipids ina bi- or multilayer with an aqueous core. Liposomes have been used toencapsulate several compounds, such as enzymes, proteins and drugswith different targets [37]. Liposomes are also the most studied NPworldwide to deliver antimicrobial drugs [37]. The foremost importantadvantage is the use of biocompatible and biodegradable constituents,which allows the use of liposomes without signicant toxicity [47,48].They are also versatile drug carriers since their physicochemical proper-ties can be easily transformed by changing the phospholipids, their pro-portion and size, their charge and even their sensitivity to externalstimuli, such as pH and temperature [47,48]. Their versatility applieseven to the drugs capable of being encapsulated, allowing theencapsulation of both hydrophilic and hydrophobic drugs and thecoencapsulation of two or more drugs [28,48]. Additionally, their simi-larity to the cell membrane allows fusion with microbes by endocytosis[34,37]. In fact, it has been shown that the probability of inducing drugresistance is lower when the basis of the antibacterial mechanism is afusion between the liposome and the bacteria [27].

In the specic case of H. pylori infection, phospholipids can also cre-ate a hydrophobic layer able to avoid bacterial attachment to themuco-sa and provide fatty acids to repair the gastricmucosa [28]. Additionally,it is possible to benet from the vacuolated protein synthesized bymostofH. pylori strains to destabilize the bilayer [24]. Further, it is possible touse phosphatidylethanolamine (PE) for selective binding to the recep-tors present on the bacterium, allowing active targeting and blockingthe adhesion of the bacteria to the gastric mucosa [3,24].

Given all the abovementioned advantages, several studies have beenperformed in order to develop a novel system for H. pylori eradicationthrough the use of liposomes (Table 1).

3.1.1. Simple liposomesSimple liposomes have been studied in order to encapsulate antibi-

otics. For instance, Bardonnet et al. (2008) designed two different lipo-somes loading metronidazole and ampicillin, consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or epikuron 170(E170) and both having cholesterol (Ch) [24]. In both formulations asynthetic glycolipid containing fucose was incorporated since somestrains of H. pylori are able to link to fucosylated Lewis b antigens

Table 1Physicochemical properties of reported liposomes and their mechanism of action associated with H. pylori eradication.

Particle compositionPhysicochemical properties of the optimized

particle Mechanism of actionRef.

(year)

Ant

ibio

tics

1. Cholesterol + DPPC

2. Cholesterol + E170 (with at least 10% of phosphatidilethanolamine(PE))

At both formulation a synthetic glycolipid was added

Size (nm) 147 > 163

Encapsulation of antimicrobial drugs(Metronidazole and Ampicillin).

Glycolipid containing fucose and PE informulations with E170 are used as a

specific ligand to H. pylori

[24] (2008)

-potential (mV) -20.0 (E170-Ch-glycolipid)> -2.9 (DPPC-Ch-glycolipid)

PDI 0.10 > 0.11%EE 4.8 (DPPC-Ch-glycolipid with

Ampicillin) > 13.9 1.0 (E170-Ch with Ampicillin)

a) Hydrogenated L--phosphatidylcholine

b) Cholesterol c) Linolenic acid

Size (nm) 88 3The liposome fuses with the bacterial

membrane, loading linolenic acid, whichpresents antibacterial activity

[27] (2012)

PDI 0.17 0.01

-potential (mV) -78 4

Vac

cine

s

a) Phosphatidylcholineb) Cholesterol

Size (nm) 100 500 Delivery of a recombinant peptidecomposed of CTB and urease B subunitepitope to induce prophylactic and

therapeutic protection

[51] (2007)%EE 71.4 before and 68.6 after 1

month of storage at 4 oC

a) Dioleyphosphatidylethanolamineb) Dimethylaminoethanecarbamolch

olesterol

c) Polyethylene glycol 2000-PE

No data availableDelivery of a multi-epitope DNA-

prime/peptide-boost vaccine to induceimmune protection

[52] (2011)

avai

: PC (7:

> 2

> 8

: PC (7:

.6

7

0.8

oxic

: 72

Sim

ple

lipos

omes

173D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186Ant

isen

seol

igon

ucle

otid

es

Cationic liposome using a commercial transfection reagent (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate)

No data

Ant

ibio

tics

Common to inner and outer liposomes:

a) Phosphatidylcholine

b)

Specific of inner liposome:

a) Stearylamine (SA)

Specific of outer liposome:

a) Phosphatidilethanolamine

Optimized formulationPC:Ch:PE

Size (m) 1.2

6.7

% EE (RBC/ innerliposome)

33

Optimized formulationPC:Ch:PE

Size (nm) 791

PDI 0.08

-potential (mV) 11 %EE Am

RBC

Cholesterol

Dou

ble

lipos

omes

PDI = polydispersity index.

EE = encapsulation efciency = ratio between the actual and theoretical amount of amoxicillable

Encapsulation of an antisenseoligonucleotide for p-50 (NF-kB dimer)in order to decrease the gastric injuries

induced by the activation of iNOS

[53] (2001)

:Ch:SA (7:3:0.1) and3:0.1)

Delivery of an antimicrobial drug(AMOX in outer liposome) and a drugwith both antimicrobial and antacid

properties (RBC in inner liposome). PE isused as a specific ligand to the bacteria

[47] (2011)

.4 (inner liposomes)

.2 (outer liposomes)

:Ch:SA (7:3:0.5) and3:0.2)

[54] (2012)

0.4

illin: 67.9 1.1

.6 1.2lin drug loaded.

174 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186through amembrane protein (BabA2) [24]. Characteristics of the NP aredescribed in Table 1. The encapsulation efciency (EE) varies among theformulations, depending on the phospholipid and on the drug, reachingthe maximum of 13.9% in the formulation with E170 and Ch [24]. De-spite the low EE, liposomes with DPPCglycolipidCh contained suf-cient antimicrobial drug to achieve an antimicrobial effect [24].Nevertheless, the authors assumed that the method can be improved[24]. Epiuorescentmicroscopy studies showed that Chmay play an im-portant role in the interaction between the bacteria and liposomes, dueto the already studied afnity ofH. pylori to this steroid [24]. The synthe-sized glycolipid demonstrated to be important in the interaction withcoccoid forms of specicH. pylori strains, namely thosewhich expressedBabA2 [24]. In spite of the promising results from the evaluation of thebacteriumliposome interaction, effectiveness studies of this interac-tion in killing thebacteria are lacking [24]. The same research groupper-formed new assays to evaluate the stability of liposomes in acidicconditions [49]. The results showed that these liposomes decrease theinternal pH to pH 4 when drastic conditions are imposed, namelypH 1.2 to 2 [49]. This fact makes this system suitable to be used to en-capsulate drugs such as amoxicillin and clarithromycin whose half-lifetime is signicantly higher at pH 4 compared to pH 2 [49]. Actually,the half-life time of these drugs are 19.0 h and 176.9 h (amoxicillin)and 1.3 h and 96.7 h (clarithromycin), at pH 2 and pH 4, respectively[49]. Additionally, agglutination assays conrmed the chemical stabilityof the synthetic glycolipid [49]. In 2012, Obonyo et al. developed a novelantibacterial nanoparticle, using linolenic acid as an antibacterial drug[27]. Amphiphilic properties of linolenic acid (LLA) allowed the incorpo-ration into the phospholipid bilayer of a liposome composed of hydro-genated L--phosphatidylcholine, cholesterol and LLA [27]. Contraryto that observed in conventional therapy, namely amoxicillin, thenovel NP killed both spiral and coccoid forms of the bacterium [27].The NP was also effective in killing all strains of H. pylori, even a strainresistant to metronidazole [27]. The bacterium did not acquire drug re-sistance when sub-bactericidal concentrations of the NP were evaluat-ed, opposed to those observed with metronidazole and free LLA [27].

Simple liposomes have also been studied as an alternative to over-come the limitations of the development of successful vaccines. Althougha prophylactic approachmay seem a powerful and economicway to con-trol the infection by H. pylori, especially in developing countries, the de-velopment of a vaccine able to cause full sterilizing immunity in animalmodels have faced several problems [5052]. In fact, pharmaceuticalcompanies have decreased their investment in this eld for the past10 years [50]. However, novel vaccination strategies have emergedusing selected antigens correlated with the pathogenesis of this infection[51]. Liposomes are an attractive delivery system because they are able toprotect the payload from the hostile gastric environmentmaking oral de-livery possible, promote a sustained release and cause immunological re-sponses [51]. In 2007, Zhao et al. used Escherichia coli to express a fusionpeptide (CtUBE) composed of a cholera toxin B subunit (CTB) and a ure-ase B linear epitope [51]. CTB was used due to its properties as a carrier,adjuvant and immunogen compound [51]. Despite a small portion of vac-cinated mice (14.3%) that developed minor or moderate gastritis, ureaseand histological tests and quantication of H. pylori colonies in mousestomach showed that vaccinated mice were signicantly protected fromH. pylori infection [51]. The nanoparticle also showed therapeutic proper-ties, promoting a signicant reduction in the load ofH. pylori in the stom-ach [51]. The increase of specic serum IgG andmucosa IgA corroboratedabovementioned results [51]. Although protection responsewas correlat-edwith Th1 lymphocyte response, further studies are necessary to under-stand the mechanism behind its prophylactic and therapeutic actions[51]. Later, Moss et al. resorted to informatic tools to design an intranasalmulti-epitope DNA-prime/peptide-boost vaccine encapsulated in lipo-somes [52]. Contrary to that observed in the administration ofH. pylori ly-sate or an empty plasmid intranasally or even the novel vaccineadministered intramuscularly, the intranasal vaccine showed therapeutic

effects [52]. As a consequence of the induced immune response, H. pyloriinfection signicantly decreased [52]. Further studies are necessary to un-derstand the enhanced effectiveness of the intranasal route [52].

In an indirect approach, liposomes have also been used to encapsu-late antisense oligonucleotides since they can improve their stabilityand intracellular delivery [53]. Previous studies showed that induciblenitrite synthase is increased in gastric mucosa of H. pylori infected pa-tients, being the production of NO responsible for gastric injuries [53].In fact, Lim et al. (2001) reported the relation between H. pylori andthe activation of an oxidant-sensitive transcription factor (nuclear fac-tor B or NF-B), which led to the induction of iNOS expression and ni-trite NO production [53]. Ultimately, it was shown that the bacteriainduce apoptosis in gastric epithelial AGS cells (human gastric adeno-carcinoma cell line) [53]. Similar to what had been observed with anti-oxidants, catalase and an inhibitor of NF-B, a liposome loadingantisense oligonucleotides for p50 (NF-B dimer) was able to inhibitthe increase of p50 and decrease iNOS expression and nitrite production[53]. Thus, apoptosis of gastric cells decrease [53].

3.1.2. Double liposomesGiven the lack of conventional liposomes, such as low entrapment

efciency, instability and unsustained release due to the possibility ofa breach in the phospholipid bilayer, double liposomes have been stud-ied [47]. They are composed of smaller liposomes inside a lipid bilayer,which protect inner liposomes against external risk [47]. Double lipo-somes have a higher drug loading capacity, higher stability and can pre-vent chemical change in free drugs, being seen as an effective deliverysystem [47,54]. However, the instability resulting from storage at hightemperatures may be more pronounced in double liposomes due totheir large size [54].

Singh et al. (2011) developed a double liposome loading ranitidinebismuth citrate (RBC) in the inner liposome and amoxicillin trihydratein the outer liposome [47]. In vitro drug release showed that after 12 h,only 32.6 1.5% of amoxicillin and 20.3 2.8% of RBC were released[47]. Although higher thanwhat had been observedwith plain amoxicil-lin + RBC, the % of growth inhibition was 86.75% [47]. Agglutination as-says revealed clumps of H. pylori when treated with double liposomes,reecting the vectorization towards the bacterium when the nanoparti-cle is functionalized with PE [47]. In the following year, Jain et al. per-formed novel assays with a new optimization of the double liposomedesigned by Singh et al. [54]. In vitro drug release studies showed asustained release of both drugs [54]. Stability studies showed an in-creased size of vesicles and a decrease in the number of vesicles/mm3

when stored at 28 C [54]. Additionally, a signicant loss of drugwas ob-served after 30 days of storing both at 4 C and 28 C [54]. Despite the in-stability under room temperature, the results of ex vivo and in vivostudies are promising. H. pylori growth inhibition was higher in thepresence of DL, compared with free amoxicillin + RBC [54]. In vivo stud-ies using albino rats supported the enhanced antisecretory and ulcer-protective action of double liposomes when compared to amoxicillin+ RBC [54].

3.2. Polymeric particles

Polymeric particles are extensively studied due to their mechanicalstability and loading capacity [3]. Additionally, it is possible to modifytheir biodistribution characteristics, resorting to the change of physico-chemical properties such as size [55]. Indeed, the surface of polymericparticles can be personalized in order to augment interactions withthe target cell and with the immune system [56]. Several polymersalso have mucoadhesive properties, which are suited to enhance theresidence time in the stomach and to overcome lower absorption of sev-eral drugs [25,55]. Polymeric particles can also protect drugs from pro-teolytic enzymes, increasing oral bioavailability [56]. Furthermore,polymers usually present several mechanisms to combat microbes,

hence it is unlikely that H. pylori would develop resistance against

them [38]. Therefore, polymers have been the subject of study for appli-cation in H. pylori eradication.

3.2.1. Polyacrylic acidPolyacrylic acid or carbopol is a mucoadhesive polymer, being used

to encase other compounds in order to increase theirmucoadhesiveness[57]. In fact, mucoadhesion to the stomach and small intestine of ratswas proven both in vitro and in vivo [57].

In 2001, Cua et al. developed an amoxicillin-loaded ion-exchangeresin encased in a polymeric microsphere [58]. The size of the micro-sphere was 133 39 m and the mass percentage of the drug relativeto the coated drug-resin complexwas 7.87 0.35% (w/w) [58]. The au-thors concluded that carbopol 934 microparticles as well as an attemptwith polycarbophil failed in signicantly prolonging retention time inthe stomach [58]. Additionally, distribution of amoxicillin-resin on themucosa was better when no-polymer was coated [58]. In 2012, Harshadeveloped an oral suspension with pure amoxicillin and amoxicillinloaded in nanospheres of 200 to 404 nm [59]. Encapsulation efciencywas 85.5 0.7% [59]. Studies of drug release demonstrated an initialburst effect, followed by a controlled release during 12 h [59]. The for-mulation was stored as dry suspension and further reconstituted withxantham gum before use [59]. At low temperatures (35 C) or atroom temperature and during 12 months, amoxicillin did not changeeither in external morphology or drug content [59].

3.2.2. ProteinsThere are several advantages in usingproteins as drug carriers (sum-

marized in Table 2), highlighting their biodegradability, non-antigenicproperties, nutritional value and the existence of rich and renewablesources [60].

Gliadin consists in a group of proteins extracted from gluten [61].Gliadin nanoparticles have been studied as a possible drug carrier forH. pylori eradication due to theirs mucoadhesive properties and theirtropism for upper gastrointestinal areas [61]. Additionally, their smallsize allows penetration into the gastric mucosa and their hydrophobic-ity permits the development of nanoparticles able to protect the antibi-otic and control its release [62,63]. Umamaheshwari and Jain (2003)used acetohydroxamic acid-loaded gliadin nanoparticles (GNP) com-bined with fucose-specic (Ulex Europaeus agglutinin I lectin UEAI) or with mannose-specic (conconavalin A lectin Con A) lectins toeradicateH. pylori [62]. In fact, lectins are known to bind to bacterial sur-face carbohydrates, allowing active targeting [62]. Additionally, pep-tides and proteins coated with lectin ensure higher protection againstdigestion and enhance uptake [60]. Studies conrmed the enhancementof afnity to pig gastric mucins and the ability to agglutinate H. pylori,contrary to that observedwithGNPalone [62]. In vitro growth inhibitionstudies demonstrated the efcacy of GNP functionalized with lectins,reaching about 95% when Con A GNP is used [62]. In situ adherencestudies demonstrated the capacity of lectins to plug the carbohydratereceptors and, subsequently, the inhibition of the attachment of the bac-teria to themucosa [62]. In the following year, the same research groupused gliadin nanoparticles to delivery amoxicillin [63]. Amoxicillin re-lease was controlled by gliadin nanoparticles, however in the presenceof pepsin (stomach enzyme) the release rate was higher due to the di-gestion of gliadin [63]. Evaluating mucoadhesion in albino rats, the au-thors found that 82% of the nanoparticles remained after 2.5 h whichcorroborates the mucoadhesiveness of gliadin nanoparticles [63]. Al-though plain amoxicillin showed faster and complete growth inhibitionin vitro, in vivo clearance demonstrated that complete eradication wasnot achieved even when using the highest dose, contrary to that

Table 2Physicochemical characteristics and mechanism of action of micro- and nanoparticles composed of proteins, more specically gliadin and gelatin, and applied to H. pylori eradication.

zed

)

NP)

func

1.3

1 1

73.7

g on

iam

xicil

175D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186Particle composition Physicochemical properties of the optimi

a) Gliadinb) Pluronic F68c) Con A or UEA I

Size (nm) 422 12 (UEAGNP

419 20 (ConA G

potential (mV) 26.6 0.8 (without % EE (w/w) 58.2 3.2

a) Gliadinb) Pluronic F68

Size (nm) 312 12

potential (mV) 26.6 0.8% Payload 61.52 2.2

% EE 66.54 3.8

a) Gliadinb) PluronicF68

Size (nm) 250 500

potential (mV) 22.8 % EE 43.7 2.3 73.1

a) Gliadinb) Pluronic F68

Size (nm) 450600

potential (mV) 22.8 % EE (maximum) CLA: 53 2.3 86.

OME: 49.6 2.8

a) Gliadinb) Pluronic F68c) Con A

Size (nm) 620 83

potential (mV) 27.46% EE AMOX: 84.6 1.34

CLA: 90.28 1.83

OME: 66.2 2.27

a) Aminated gelatinb) Glutaraldehyde

Size (m) 46 > 55 (dependinglutaraldehyde)

a) GelatinSize (nm) 501000 (average d

89.2 0.5% drug content

Prot

eins

to

deliv

er a

ntib

ioti

cs

EE = encapsulation efciency = ratio between the actual and theoretical amount of amoPayload = encapsulated drug/gliadin nanoparticle yield.

Drug content = weight of drug in nanoparticle/total weight of the nanoparticle.particle Mechanism of actionRef.

(year)

Carrying of acetohydroxamic acidto eradicate H. pylori using activetargeting

[62](2003)tionalization)

Delivery of amoxicillin usingmucoadhesiveness of gliadinnanoparticles

[63](2004)

Varyingdrug: gliadin

ratio

Delivery of clarithromycin usingmucoadhesiveness of gliadinnanoparticles

[64](2006)

Encapsulation of clarithromycin(antibacterial properties) andomeprazole (antacid properties)using gliadin as a mucoadhesivecomponent

[61](2008)

Varyingdrug: gliadin

ratio.3

3.9

Encapsulation of a triple therapy(amoxicillin, clarithromycin andomeprazole) using gliadin as amucoadhesive component andlectin as a specific binding

[65](2008)

the % of Delivery of amoxicillin usingmucoadhesiveness of aminatedgelatin

[66](2000)

eter was 571 nm) Immediate and sustained release ofamoxicillin

[67](2013)

lin drug loaded.

observed with nanoparticles [63]. In 2006, they tried to use gliadinnanoparticles to encapsulate clarithromycin [64]. These nanoparticlesproved their mucoadhesiveness through in vitro and in vivo studies,being suitable for localized delivery, especially to the upper region ofthe stomach [64].

In 2008, Ramteke and Jain tested clarithromycin- and omeprazole-containing gliadin nanoparticles [61]. A sustained release andmucoadhesiveness were proved through in vitro and in vivo methodsand a synergic effect in H. pylori growth inhibition was observed whenclarithromycin-NP and omeprazole-NP were used simultaneously [61].This synergic effect may be due to the improvement of antibacterial ac-tivity of clarithromycin at higher pH [61]. Although higher than that ofthe plain drugs, % of growth inhibition of both NPs combined was only83.7% [61]. The same research group (2008) developed lectin-conjugated gliadin nanoparticles in order to deliver a triple and synergictherapy (amoxicillin, clarithromycin and omeprazole) [65]. When com-pared with gliadin nanoparticles, lectin-conjugated gliadin nanoparti-cles revealed an enhancement in in vitro antibacterial studies andin vivo clearance, reaching 94.83% and 83.3% of eradication and clear-ance rate, respectively [65]. Similarly, in vitro and in vivo studies ofmucoadhesive properties showed a slight increase of adhesion to themucosa when the nanoparticles were coated with lectin [65].

Gelatin has also been studied due to its long history of security andsafety, being used in pharmaceuticals, cosmetics and food, based on itsFood and Drug Administration (FDA) classication as GRAS (generallyregarded as safe) [60]. Wang et al. (2000) tested aminated gelatin mi-crospheres due to the possibility of a positive charge increase in electro-static attraction to themucosa [66]. Several parameters, such as the % of

glutaraldehyde, the time of cross-linking reaction and the higher pH,contributed to a sustained release [66]. Modied gelatin microspheresdemonstrated a slower release comparativelywithmicrospheres of reg-ular gelatin with the same % of glutaraldehyde [66]. In vivo studiesproved that aminated gelatin microspheres have a higher gastric reten-tion time, however further studies are necessary to understand themechanism behind mucoadhesiveness of these microspheres [66].

Harnessing the mucoadhesiveness of gelatin, Harsha (2013) de-signed a suspension for immediate and sustained release of amoxicillinusing gelatin nanoparticles [67]. Amoxicillin-loaded gelatin micro-spheres were added to a suspension containing xantham gum, D-sorbitol powder and citric acid asmajor components [67]. Nanoparticleswere able to induce a sustained and controlled release of amoxicillinduring 12 h and were stable for 24 months even under hostile condi-tions (25 C and 60% humidity) [67]. Supplementary studies are neces-sary to conrm their effectiveness in H. pylori eradication [67].

3.2.3. PolysaccharidesPolymeric carbohydrate molecules, named polysaccharides, such as

chitosan and alginate, have also been used against H. pylori (Table 3).Chitosan is a linear and cationic polymer, composed of D-glucosamine

and obtained from chitin deacetylation [68]. It has been suggested as apromisingdrug carrier owing to itsmucoadhesiveness andbiocompatibil-ity [25]. Since it is positively charged, it can interact with sialic acid resi-dues of mucin in the stomach which present a negative charge [69]. Italso may be useful for drug release in response to pH decrease, due toswelling of chitosan microspheres in acidic medium [69]. Furthermore,several reports proved a broad-spectrum antimicrobial effect of chitosan

Table 3Physicochemical properties and mechanism of action of particles composed of polysaccharides, namely chitosan and alginate, applied to eradication of H. pylori.

Particle composition Physicochemical properties Mechanism of actionRef.

(year)

a) Chitosan (87% degree of deacetylation (DA))

Amoxicillin MetronidazoleLocal amoxicillin and

metronidazole delivery[73]

(1999)Size (m) 50 50%EE 81.2 4.5 99.4 6.9

n a

en

A

0)

(p

dinlink

A

icil

176 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186a) Chitosan (>80% DA)

AmoxicillinSize (m) ~10%EE Close to 100

a) Chitosan (85% DA)b) Glutaraldehyde

Size (m) 50.4 98.2

%EE 38 70

a) Chitosan (84%DA)Size (m) 2.5

%EE 76

a)

Size (m) 2.0-3.0

-potential (mV) 26.68 %EE 8.04 0.00 whe

69.37 5.84 wh

a) Chitosan (88.5% DA)

Or

a) Chitosan (95% DA)

88.5% D

Size (nm) 96.12 (70 13

PDI 0.16 0.03

-potential (mV) 23 (pH 2) -10a) Chitosan (15% DA)b) Genipinc) Sodium triphosphate

pentabasic

Size (m) 170 15

-potential (mV) -6 -> 30 (Depenperiods of cross

a) Hidrophobically modified alginate

Size (m) 9

%EE 65-70

Poly

sacc

hari

des

Chit

osan

to

deliv

er a

ntib

ioti

csA

lgin

ate

and

vacc

ines

Chit

osan

and

its

anti

bact

eria

l eff

ect

Chitosan (87% DA)

EE = encapsulation efciency = ratio between the actual and theoretical amount of amox

PDI = polydispersity index.Metronidazole Delivery of amoxicillin ormetronidazole usingmucoadhesiveness of

chitosan

[74](2002)

~1029 60 (varying withreacetylation time)

Delivery of amoxicillinthrough encapsulation in amucoadhesive microsphere

[25](2007)and stirring speed

Encapsulation of amoxicillinin a mucoadhesive

microsphere

[77](2012)

Delivery of tetracyclinethrough encapsulation in a

mucoadhesive microspheres

[69](2002)dded before cross-linking

added to preformed microspheres

95% DAUse antibacterial propertiesof chitosan to eradicate H.

pylori

[41](2009)

96.16 (71 129)

0.18 0.05

H 7) 25 (pH 2) -8 (pH 7)

Use antibacterial andmucoadhesive properties ofchitosan to remove bacteria

from the stomach ofinfected people

[44,68](2013)g on pH, crosslinking and on time

ing)

Encapsulation of H. pyloriurease in order to promote

in vivo immunization

[82](2004)

t different polymer:drug ratio

lin drug loaded.

177D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186[41,70]. A serious disadvantage is its higher solubility at acidic pH [68].Nevertheless, this problem has been overcome by resorting to acrosslinking agent, such as glutaraldehyde or genipin [68]. Genipin,which is a naturally occurring cross-linking agent, presents lower cell tox-icity compared to glutaraldehyde and can inhibit H. pylori colonization[71,72].

Several studies have beenperformedbasedupon the abovementionedin order to encapsulate antibiotics. For instance, Shah et al. (1999) devel-oped chitosan microspheres in order to deliver amoxicillin and metroni-dazole to the site of infection [73]. However, a sustained release intosimulated gastric uid (pH 1.2) was not achieved, since chitosan micro-spheres were highly porous, which led to the release of amoxicillin andmetronidazole in 2 h [73]. In 2002, Portero et al. used reacetylated chito-san microspheres to encapsulate amoxicillin and metronidazole sinceprevious work had proven the interaction between these microspheresand the mucosa and the sustained delivery of amoxicillin [74,75].Reacetylation was used to reduce chitosan solubility and its inuenceon in vitro release and antimicrobial properties of drugs was evaluated[74]. Reacetylation affected the encapsulation of metronidazole, reducingthe encapsulation efciency [74]. Antimicrobial activity depends on thereacetylation time, however if short reacetylation time is used, a sustainedrelease is achieved without loss of antimicrobial properties [74]. Chitosanmicrospheres were also used by Patel et al. (2007) to encapsulate amox-icillin and the inuence of different variables was tested [25].Mucoadhesion decreased with the increase of stirring speed and withthe decrease of polymer:drug ratio [25]. Stirring speed also had a negativeeffect on drug entrapment efciency [25]. In vitromucoadhesive tests alsoshowed that some microspheres adhered even after 12 h [25]. Releasestudies demonstrated that amoxicillin was released more promptly inacidic pH than in basic pH [25]. In vivo studies revealed higher H. pyloriclearance when microspheres were used compared to that observedwith plain amoxicillin [25]. In 2010, Raval et al. characterized spraydried microspheres of amoxicillin [76]. Physicochemical and morpholog-ical studies revealed that by increasing glutaraldehyde concentration andthe duration of crosslinking, the ability to swell decreases [76]. Addition-ally, the percentage of swelling is correlated with in vitro drug release,hence it may be possible to control permeability to solutes adjustingabovementioned parameters [76]. In 2012, Patel and Patil tested the syn-thesis of amoxicillin mucoadhesive microparticles using supercritical CO2as an alternative to conventional processing methods [77]. Application ofthis supercritical uid technology to the development of novel micropar-ticles was successful [77]. High mucoadhesion and a sustained release atboth pH1.2 and 7.8was achieved [77]. In vivo studieswith administrationtwice a day for three consecutive days revealed a higher antimicrobial ef-fect of amoxicillin-loaded microparticles when compared to powderamoxicillin [77].

Tetracycline had also been encapsulated in chitosanmicrospheres de-spite being more stable than amoxicillin in acidic medium [69]. The rstreport was published by Hejazi et al. in 2002 [69]. Tetracycline-microspheres were dissolved at pH 1.2 and 2.0, leading to an abrupt andrapid release of the drug [69]. Although more gradual, the release atpH3.0 and 5.0was similar, releasing almost 70% after only 3 h [69]. Hejaziand Amiji continued this research and published in 2003 a novel reportwhere they studied the gastric residence time of tetracycline-loading chi-tosan microspheres in gerbils using radioiodinated [125I] chitosanmicrospheres and tritiated-[3H]-tetracycline [78]. The results were disap-pointing, showing a similar retention prole between the tetracyclineloaded and the plain tetracycline in aqueous solution [78]. The main con-clusion of this study is that chitosan microspheres prepared by ioniccross-linking are not suitable if the aim is a longer residence time [78].In this context, Hejazi et al. (2004) studied the inuence of crosslinkingon the gastric residence, obtaining higher residence times with micro-spheres produced by chemical crosslinking as opposed to those producedby ionic precipitation [79]. A review of their completeworkwas also pub-lished [80]. In vivo studies showed that although more efcacious than

plain tetracycline, tetracycline-loaded chitosan microspheres revealedlower reduction of the levels of the bacteria as well as the serum gastrinlevels in comparison to a triple therapy (lansoprazole, amoxicillin andclarithromycin) [80].

Chitosanhas also been used for a different purpose. Its bactericide ef-fect and mucoadhesiveness may enable the use of particles of chitosanto adhere, kill and removeH. pylori from the stomach.With this purposein mind, Luo et al. (2009) studied the antibacterial effect of nanoparti-cles of chitosan [41]. This research group performed in vitro andin vivo studies to conrm that chitosan nanoparticles present higheranti-H. pylori efcacy than chitosan powder [41]. In 2013, Nogueiraet al. studied the inuence of the gastric medium in the effectivenessof chitosan microspheres mimicked through an ultrathin chitosan lm[70]. Chitosan was considered suitable for gastrointestinal use atpH 2.6, 4 and 6 and independent of the presence of urea [70]. However,adhesion of H. pylori to chitosan lms was lower at higher pH and wasremarkably reduced in the presence of pepsin [70]. Similarly, pepsin re-duced the death of chitosan-adherent H. pylori [70]. Nevertheless, theantibacterial effect of chitosan was proven, reaching death of 93% atpH 2 and more than 75% of the bacteria at pH 6, when urea and pepsinwere absent [70]. In the same context, Fernandes et al. studied the char-acteristics of genipin-stabilized chitosan microspheres under acidic pH[44]. In order to achieve stability in acidic mediumwithout diminishingthe microspheres' ability to link to mucins of the mucosa, crosslinkingwas performed using 10 mM of genipin and during 1 h [44]. Despiteswelling to twice their size, these microspheres were stable during7 days in SGF and revealed the ability to remain in the stomach during2 h [44]. Further studies were performed, revealing the ability to binddifferent strains of H. pylori independently of pH and of the presenceof pepsin, contrary to that observed in previous studies where Nogueiraet al. used an ultrathin chitosan lm [68]. These novel studies demon-strated microsphere efciency in reducing the attachment betweenthe bacteria and gastric cells in 5076% and in 4756% when addedafter and before H. pylori-gastric cell pre-incubation, respectively [68].Although reducing 20% of cell metabolic activity, these microsphereswere not considered cytotoxic according to ISO international standard10993-5 [68]. Nonetheless, additional studies are necessary to verify ifrepeated treatments are sufcient to complete eradication of H. pylori[68].

Alginate is also a promising option for drug delivery due to its bio-compatibility, low toxicity and inexpensiveness [81]. A hydrophobicallymodied alginate-based microparticle was developed by Leonard et al.(2004) with the purpose of creating a vaccine against H. pylori [82]. Hy-drophobic interactions allowed good retention of proteinswhich are re-leased upon addition of a surfactant or by hydrolysis by lipases [82].Despitemucoadhesiveness of thesemicroparticles, subcutaneous vacci-nation of mice obtained more promising results when compared withoral and nasal routes [82]. However, no statistically relevant conclusionscan be deduced from these preliminary studies [82].

3.2.4. CopolymersA few studies have been performed in order to study physicochem-

ical properties of micro- and nanoparticles composed of a mixture ofpolymers with no loaded drug. For instance, in 2003, Miyazaki et al. de-veloped microspheres composed of cellulose acetate butyrate and dex-tran derivatives and evaluated their mucoadhesiveness and retentiontime in the stomach [83]. Dextrans, which are polysaccharides usedworldwide in the medical eld, were able to increase mucoadhesiveproperties both in vitro and in vivo [83]. In 2009, Lin et al. resorted to acombination of the benets of chitosan referred to in Section 3.2.3with the advantages of heparin, namely its ability to heal gastric ulcers,to develop a particle with about 130 nm [84]. These nanoparticles werepH-sensitive through the ionization of chitosan and heparin at pH 1.26.5, resulting in a polyelectrolyte complex [84]. At pH 7.0, chitosan be-comes deprotonated leading to the disintegration of the nanoparticles[84]. Fluorescence studies using AGS cells andmouse gastric epithelium

showed the adhesion and uptake of nanoparticles by gastric cells and, as

pose

178 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186Table 4Physicochemical characteristics and mechanism of action of micro- and nanoparticles coma result, degradation of nanoparticles by lysosomes [84]. These nanopar-ticles were also able to interact locally with sites of H. pylori infection[84].

Particle composition Physicochemical properties of the

a) Carboxyvinyl polymerb) Curdlan

Size (m) 250-335

a) Polyethylcyanoacrylateb) Pluronic F68c) PEG(Different

formulations were testedvarying the molecular weight of PEG

PEG 600 PEG 200

Size (nm) 280 8 230 15PDI 0.60 0.40

-potential (mV) -10.9 1.5 -7.8 1%drug (w/w) 3.5 0.11 7.5 0.2

a) Cholestyramine

b) Cellulose acetate butyrate Size (m) 20 2

a) Poly(acrylic acid)b) Poly(vinyl pyrrolidone)

AmoxicillinSize (m) 62.7 4.7

% EE 57.5 3.5

a) Carbopol-934Pb) Ethyl cellulose

Size (m) 400-1000

Size (m) 86.89 12.45 129.72 1

%EE 48.35 2.41 78.25 2.6Size (m) 109%EE 56

a) Ethylcelluloseb) Concanavalin-Ac) Chitosand) Polyvinyl alcohol

Size (m) 144.35 22

-potential (mV) 7.56 0.7%EE 72.13 1.4

a) Eudragit S100b) Polyvinyl alcohol (PVA)c) Concanavalin-A

Size (m) 188.18 2.46

-potential (mV) 18.7 0.38%EE 70.22 0.14

a) Sodium alginateb) Carbopol 934Pc) Polycarbophil

Size (m) 208.5 408.5

%EE 35.249 4.623 95 2.8

a) Chitosanb) Glutamic Acidc) -l-fucose

Size (nm) 874.97 25.49

% EE 88.5 2.8 (Amoxicillin) 91.1 2.3 (Clarithromycin58.4 3.7 (Metronidazole

a) Chitosan (85% DA)b) Y-PGA

Size (nm) 149.6 6.3

-potential (mV) 18.9 3.1%EE 23.5 2.7

a) NIPASM b) AA

c) HEM d) BPO

e) TEGDMA

Size (nm) 65 158

%EE 70.2 91.4

a) Eudragit RL 100b) Carbopol-974P

Size (m) 155-306

%EE 82 1.13 90 1.67a) Sodium alginateb) HPMC K4M

Or b) Carbopol-974 P

Size (m) 602 1.03 784 5.11

%EE 66 1.88 93 2.02

a) Eudragit RL 100b) Carbopol 934Pc) HPMC K4M

Size (m) 224 358

%EE 80 1.25 92 2.20

a) Eudragit RS 100b) Carbopol-974Pc) KPMC K4M

Size (m) 123 8.35 524 11.54

%EE 56.71 1.66 89 3.11

Size (m) 118.5 6.51 > 493.3 11

%EE 52.62 0.72 87.97 0.8

Copo

lym

ers

to d

eliv

er a

ntib

ioti

cs

PDI = polydispersity index.%drug (w/w) = percentage of the drug amount contained in 100 mg of dried material.EE = encapsulation efciency = ratio between the actual and theoretical amount of amoxicil% loading = weight of drug in SPs / total weight of the SPs.d of polymer mixture to be applied to H. pylori eradication.

Ref.A mixture of polymers has also been applied to encapsulate antibi-otics (Table 4). Nagahara et al. (1998) developed an amoxicillin-loaded mucoadhesive microsphere using carboxyvinyl polymers and

optimized particle Mechanism of action (year)

Delivery of amoxicillin usingmucoadhesiveness

[85](1998)

0 PEG 4000

Encapsulation of amoxicillin to drugdelivery, resorting to mucoadhesive

properties of polymers

[55](2001)

220 100.28

.3 -5.1 1.11 8.1 0.23

Encapsulation of AHA an ureaseinhibitor, to local delivery

[86](2003)

Clarithromycin Delivery of amoxicillin orclarithromycin using mucoadhesive

microspheres

[87](2005)65.4 4.6

93.5 5.7

Delivery of amoxicillin[88]

(2005)

3.87Delivery of clarithromycin

[89](2008)5

Delivery of amoxicillin[90]

(2009)

Use functionalized microspheres fora controlled and local delivery of

clarithromycin

[91](2008)

Use Con-A microspheres to deliveramoxicillin

[92](2014)

Encapsulation of clarithromycin intomucoadhesive microspheres

[93](2009)35

Use affinity of H. pylori receptors tofucose and use of mucoadhesiveness

of polymers to create a triple drugdelivery system

[94](2009))

)

Delivery of amoxicillin, usingmucoadhesive nanoparticles

[95](2010)

Varying Encapsulation of amoxicillin to drugdelivery, resorting to mucoadhesive

properties of polymers

[97](2010)composition

Varying

composition

Varying

composition

Varying

Use polymer mucoadhesiveness todeliver loaded clarithromycin to

localized action

[98](2010)

compositionVarying

[43](2011)composition

Use polymer mucoadhesiveness todeliver loaded furazolidone to

localized action

[100](2010)

Use polymer mucoadhesiveness todeliver loaded amoxicillin

[101,102]

(2010/2011)

.23 Use polymer mucoadhesiveness todeliver loaded clarithromycin

[103](2012)3

Varying

composition

Varying

composition

Varying

composition

lin drug loaded.

179D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186a) Ethylcelluloseb) Eudragit EPOc) Poly(vinyl alcohol)d) Glycerol monooleate

Size (m) 500-1000

%loading 6.82

a) Polycationic chitosanb) Polyanionic alginatec) Pluronic F-127

Size (nm) 651

-potential (mV) 59.76% EE 91.23

a) Sodium alginateb) Carbopol-934Pc) Calcium chloride di-

hydrate

Size (m) 890 2.8 980 3.2

%EE 68.2 2.5 78.0 2.1

a) Chitosan (85% DA)

b)Heparin(aqueous phase of a water-in-oil emulsification)

Size (nm) 296.5 6.3

-potential (mV) 29.8 3.1%EE 54.3 2.8

% loading 19.2 1.2

a) Sodium alginateb) Sodium carboxymethy

cellulose

c) Magnesium aluminium silicate

d) Chitosan

Size (m) 745 889

%EE 52 0.78 92 1.2

% loading 5.2 0.78 11.0 0.8

a) Sodium alginate Size (m) 17 1

Particle composition Physicochemical properties of the

Table 4 (continued)curdlan [85]. Stomach residence time and in vivo clearance were en-hanced three and ten times, respectively, compared to plain amoxicillinsuspension [85]. In 2001, Fontana et al. took into accountpolyethylenoglycol (PEG) properties, specically their ability to avoidmacrophage opsonization and, consequently, escape from the immunesystem, in order to develop a novel nanoparticle for amoxicillin delivery[55]. Experimental studies showed PEG inuence on particle size, po-tential zeta and drug entrapment and proved that PEG coating certainlyreduced the opsonization by macrophages [55]. PEG was also responsi-ble for the reduction of drug release in human plasma, conversely tothat observed in drug release assays at pH 7.4 [55]. The release of amox-icillin followed a biphasic prole, being rapidly released at the rst stage[55]. Experimental studies also evidenced the effect of urease in increas-ing amoxicillin release owing to NP degradation [55].

A mixture of cholestyramine and cellulose acetate butyrate was alsoused to create a drug delivery system by Umamaheshwari et al. (2003)[86]. Cholestyramine is a mucoadhesive polymer which may locally in-teract with gastric mucosa through electrostatic forces [86]. Additional-ly, it can be used to encapsulate any drug of anionic species [86].Acetohydroxamic acid release was higher at pH 1.2 than at pH 7.4[86]. Additionally, the developed microparticles revealed bothmucoadhesiveness and oating properties, being suitable for enhancingthe retention time in the stomach [86]. Further studies are necessary toprove their efciency in killing H. pylori [86].

In order to reduce water solubility of poly(acrylic acid), a well-known mucoadhesive and biocompatible polymer, Chun et al. (2005)

b) Pectinc) Calcium chlorided) Ethylcellulose

%EE 83 1.3

a) Chitosan (85% DA)b) Heparinc) Genipind) Fucosee) Sodium cyanoborohydride

Size (nm) 249.6 4.2

-potential (mV) 27.2 1.6 % EE 48.7 2.8

a) Eudragit RS

Size (m) 1.1283 0.0551 9.9936

PDI 0.141 0.029 0.294 0

-potential (mV) 44.25 1.08 65.39 2.315.77 3.44 31.72 3.5

%EE 99.47 2.06 100.10 3

%Loading 8.07 1.14 25.79 2.06Use mucoadhesiveness of GMO todeliver psoralen, a linear

furanocoumarin compound

[104](2011)

Delivery of an antimicrobial agent,namely amoxicillin resorting to

mucoadhesiveness andmucopenetrating properties

[35](2011)

Use mucoadhesiveness of polymersto vectorize clarithromycin to

stomach

[105](2011)composition

Delivery of amoxicillin usingadvantages of chitosan and heparin

[106](2012)

Use coat of chitosan to vectorizeamoxicillin to the stomach

[107](2012)

Varying

composition

Varying

optimized particle Mechanism of actionRef.

(year)developed a complex microsphere adding poly(vinyl pyrrolidone)[87]. In fact, the dissolution of the complex was signicantly di-minished when compared to PVP alone and was expressivelyslower at pH 2.0 than at pH 6.8, being useful for gastric delivery[87]. While the release of amoxicillin was almost independent ofthe pH, resulting from diffusion mechanisms, clarithromycin re-lease varied with pH and resulted from the dissolution of PAA/PVP matrix [87].

In 2005, Liu et al. used a nanoparticle composed of ethylcellulosecoated with carbopol-934P to encapsulate amoxicillin [88]. Addingmucoadhesiveness and biodegradable properties of carbopol to amatrixpolymer composed of ethyl cellulose, a nanoparticle with afnity tomucosa was obtained, which was proved through in vitro and in vivomucoadhesiveness evaluation [88]. Preliminary studies of in vivoH. pylori clearance were very promising, showing complete clearanceat multidosage administration (twice a day, during three consecutivedays) [88]. Three years later, the same idea was applied to design aclarithromycin-loaded ethyl cellulose-carbopol 934P microparticle byRajinikanth et al. [89]. Microparticles manifested bioadhesiveness andin vitro oating during 20 h [89]. As a result, higher H. pylori clearancerates were obtained in Mongolian gerbils infected compared to thoseof a suspension of clarithromycin [89]. In 2009, Patel and Chavda tookthe previous idea of amoxicillin-loaded ethyl cellulose-carbopol 924Pand synthesized it with different proportions and solvents [90]. Onceagain, increased retention time in stomach was obtained due tomucoadhesiveness [90]. In vivo H. pylori clearance tests revealed no

Use a blend polymeric matrix tocontrolled release of clarithromycin

[108](2013)

Active and passive targeting ofamoxicillin using fucose and pH-

sensibility, respectively

[71](2013)

0.0921

Sustained release of metronidazole[109]

(2014)

.040

9 (SGF)

5 (water)

.21

composition

Varying

180 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186bacteria colony nor urease test positive in rats after administration ofmicrospheres twice a day, during three consecutive days [90].

Jain and Jangdey (2008) took advantage of the afnity of Con A to dif-ferent cells, via carbohydrate portions, and created a functionalizedchitosan-ethylcellulose microsphere [91]. Mucoadhesive studies in vivowere performed and revealed the gastroretentive behavior of the devel-oped formulation [91]. Additionally, the release of clarithromycin wassustained and controlled in simulated gastric uids [91]. The same re-search group used Con A conjugated with microspheres of EudragitS100 and PVA to encapsulate amoxicillin [92]. The proposedformulation showed mucoadhesive properties and a sustained re-lease during 24 h at pH 1.2 [92]. Future perspectives include in vivostudies to evaluate H. pylori eradication [92].

In 2009, Thorat et al. reported the design of mucoadhesive micro-spheres in order to load clarithromycin, accompanied by a 32-full facto-rial [93]. In vitro studies revealed good mucoadhesiveness and asustained release of the drug [93]. In the same year, Ramteke et al. de-veloped a chitosanglutamic acid nanoparticle for triple delivery(amoxicillin, clarithromycin and omeprazole) [94]. Once again, afnityof the bacteria to fucose was used as active targeting [94]. The devel-oped nanoparticle achieved a remarkable 97% eradication rate, superiorto that of nonconjugated nanoparticles and plain triple therapy [94]. Acomplete eradication was obtained in vivo, using Swiss albino mice,and histopathological studies revealed absence of infection [94]. Withthe same basis, Chang et al. (2010) designed amoxicillin-loadedchitosan/poly- -glutamic acid ( -PGA) nanoparticles, carrying a pH-sensitive hydrogel to overcome instability in the acidic medium [95].-PGA is a biocompatible polymer which can form a gel by electrostaticinteractions with chitosan [96]. Subsequent to adhesion to the gastricmucosa, calcium-alginate-gelatin hydrogels will swell and collapse,leading to the release of nanoparticles and inltration into the mucus[95]. It was evident that at pH 7.0 chitosan/ -PGA nanoparticles swelledand disintegrated, allowing rapid release of amoxicillin [95]. Confocallaser scanning microscopy revealed the location of the novel nanoparti-cle at the sites of H. pylori infection (intercellular spaces and cell cyto-plasm) [95].

Another copolymer was synthesized by Moogooee and his researchteam through the crosslinking of N-isopropylacrylamide (NIPASM),acrylic acid (AA) and hydroxyethyl methacrylate (HEM) using benzoylperoxide (BPO) as an initiator and triethyleneglycol dimethacrylate(TEGDMA) as a crosslinking agent [97]. The release of amoxicillin washigher at pH 1.0 than at pH 7.4 [97]. A sustained and controlled releasewas achievedwith smaller nanoparticles but they present lower loadingcapacity [97]. In vivo studies performed on gastric tissues of rats showedan enhancement of gastric concentration of amoxicillin compared withplain amoxicillin [97].

Still in 2010, Venkateswaramurthy et al. developed clarithromycin-loaded microparticles with Eudragit RL100 as matrix and addingCarbopol 974P, acrylic and methacrylic-based polymers, respectively, toensure mucoadhesive properties [98]. In vitro studies revealedbioadhesive properties, which might be reected in an enhancement ofthe retention time in the stomach [98]. In the next year, anotherclarithromycin-loaded mucoadhesive microsphere was tested andbioadhesive properties were also proved through in vitro studies [43].At gastric pH, carbopol 934P forms a slightly viscous gel, which inu-ences drug release [99]. Hence, in order to improve mucoadhesive prop-erties and the sustained release of the drug, this research group tried amixture of carbopol 924P and hydroxy propyl methyl cellulose (HPMC)K4M, using Eudragit RL 100 or Eudragit RS 100 to disperse the polymers[99]. This novel composition was applied to encapsulate furazolidone(2010) [100], amoxicillin (20102011) [101,102] and clarithromycin(2012) [103]. In 2013, Venkateswaramurthy et al. administered twicedaily for three consecutive days a mixture of amoxicillin-loaded MPsand clarithromycin-loaded MPs to male Wistar rats [99]. A completeeradication was achieved contrary to that observed with a solution of

plain drugs (amoxicillin + clarithromycin) [99].Liu et al. (2011) joined amixture of polymers asmatrix with a coat ofa bioadhesive polymer, namely glycerol monooleate, in order to create aoating andmucoadhesive system [104]. Glycerol monooleate was usedowing to its ability to forman in situ liquid crystal phase, characterized byhigh viscosity and bioadhesiveness in vivo [104]. Additionally, it is anFDA-approved additive due to its non-toxicity [104]. Drug releasestudies revealed a pH-dependency, being higher at acidic pH [104].Mucoadhesiveness and buoyancy were proven both in vitro and in vivoand pharmacokinetics analysis of the drug revealed and enhancementof the plasmatic half-life time [104]. In an endeavor to improvemucoadhesive particles, Arora et al. (2011) developed a nanoparticlealso able to penetrate into the gastric mucosa [35]. To achieve this aim,it was necessary to modify the charge of the chitosan surface in orderto decrease mucoadhesiveness and decrease particle size to less thanthe mesh size of mucin bers [35]. In vivomucopenetration studies re-vealed the permanency of NPs during 6 h in the deeper layers of themu-cosa, near epithelial cells [35]. Still in 2011, Pal et al. developed apolymeric mucoadhesive microsphere in order to encapsulateclarithromycin [105]. Mucoadhesiveness was proven by in vivo studies,with 72% remaining after 5 h and 37% after 7 h [105]. In vitro antibacterialeffect was lower than 25% of growth inhibition after 8 h [105]. Studies ofstabilitywere also performed, revealing higher stability at lower temper-atures with no degradation after 30 days at 48 C [105].

In 2012, Lin et al. tried to improve the challenging encapsulation oflow molecular weight hydrophilic drugs in nanoparticles composed ofchitosan and heparin usingwater-in-oil emulsication [106]. The devel-oped system presented a sustained release of amoxicillin at pH 1.2, col-lapsing at pH7.0 [106]. This evidence is important since the surroundingpH of the bacteria is neutralized by urease secretion [106]. The resultsalso showed a specic interaction between the nanoparticle and AGScell monolayers infected with the bacteria [106]. Although incomplete,theNPs revealed amore completeH. pylori eradication than plain amox-icillin [106]. Still with the purpose to increase the entrapment efciencyof water soluble drugs and to modulate their release, Angadi et al.(2012) developed microbeads of well-known polymers coated withchitosan to enhance mucoadhesiveness [107]. In fact, the compositionof the polymermatrix was decisive to obtain higher %EE, more preciselyhigher amounts of sodium carboxymethy cellulose andmagnesium alu-minium silicate resulted in higher %EE [107]. These microspheres werealso efcient in obtaining controlled release of amoxicillin [107].

Taking into account the advantages of a blend of polysaccharidema-trix, namely their worldwide use, their exibility in management ofdrug-release prole and cost effectiveness, Tripathi (2013) used sodiumalginate and pectin to create a pH sensitivemicroparticle [108]. This for-mulation showed favorable results both in vitro and in vivo, with amax-imum percentage of growth inhibition of 85% and a more effectiveclearance than plain clarithromycin [108].

Appending mechanical stability conferred by genipin and activetargeting provided by fucose, a genipin-cross-linked fucose-chitosan/heparin nanoparticle was developed [71]. According to what had beenexpected, at pH 1.2 amoxicillin was controllably released while atpH 7.0 the NP collapsed leading to a rapid release [71]. Although witha higher degree of contact with H. pylori due to its positively chargedsurface, the developed nanoparticle merely achieved 53.5 6.3 ofgrowth inhibition [71]. In vivo H. pylori growth inhibition studies andhistological examinations revealed an enhancement of the clearanceand a decrease of the inammation [71]. Similar to what had been ob-served in the previous study, these NPs were able to locally interactwith the sites of H. pylori infection, namely cellcell junctions and cellcytoplasm, reducing the disruption of cellcell junction protein [71].The effect of the NP on bacteria morphology was also evaluated, show-ing changes from a helical to a coccoid form [71].

The most recent study dated from 2014, in which a porousEudragit RS microparticle fabricated via electrospray method was an-alyzed [109]. In vitro release studies revealed a sustained release, which

may be benecial when added to the ability to be retained in the

ture

181D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186Table 5Physicochemical characteristics and mechanism of action of particles composed of a mixthrough immunization (vaccines).stomach for 8 h [109]. In vivo clearance studies support the effectivenessof this novel formulation, with a more complete clearance of H. pyloriand lower gastric inammation in H. pylori-infected mice models thanwith a pure drug solution [109].

A mixture of polymers has also been applied to the encapsulation ofprobiotics (Table 5). For example, Ko et al. (2011) developed a micro-sphere to encapsulate Lactobacillus casei ATCC 393 [96]. These organ-isms are used as probiotics, being able to produce some compoundinhibitors of toxic activity of some pathogen organisms, namelyH. pylori [96]. Additionally, it can manage side-effects of the currenttherapy which may result from qualitative and quantitative changes ofgastric microora [26]. At pH 1.5 a signicant amount of the probioticwas released after 60 min due to the instability of the microsphere atacidic pH [96]. H. pylori growth inhibition and anti-adhesive effect ofLactobacilluswere sustained during 60min even under pH 1.5, whereasa non-encapsulated probiotic presented a higher initial efcacywithoutmaintaining the effect during the 60 min [96].

The use of phytomedicine to eradicate H. pylori and the possibility touse micro- and nanotechnology to overcome its limitations have alsobeen studied (Table 5). Berberine, a plant alkaloid compound with theability to inhibit H. pylori growth and protect gastric mucosa, has alsobeen the target of study [110]. The strategy of combining chitosan and

EE = encapsulation efciency = ratio between the actual and theoretical amount of amoxicilPDI = polydispersity index.of polymers in order to treat H. pylori infections (probiotic, phytomedicine, antacids) orheparin was applied by Chang et al. (2011) to carry berberine [110]. Invitro studies proved the dose-dependence ofH. pylori growth inhibition,reaching around 53% with 12 mg L1 of berberine [110]. The novel NPshowed a signicant efcacy in increasing AGS cell viability after pre-incubation with H. pylori [110]. Further studies are required for in vivoapplication of this NP combined with other antibiotics [110]. Since chi-tosan microspheres are insufcient to obtain a controlled release of hy-drophilic drugs, a novel strategy was chosen by Zhu et al. (2012) [111].The usual strategies pursued resulted in the reduction of exibility andthe amount of amino groups, hence a loss of the mucoadhesivenessproperties of chitosan [111]. Chitosan microspheres with multipleEudragitL100 cores were chosen by Zhu et al. (2012) to encapsulateberberine [111]. In vitro release studies revealed a controlled release ofberberine, maintaining the mucoadhesiveness properties of chitosan[111]. In the following year, Pan-in et al. used amixture of ethylcelluloseand methylcellulose to encapsulate Garcinia mangostana extract (GME)[112]. GMEwith 56% byweight of-mangostin was used due to severalpharmacological effects, highlighting antioxidant, anti-inammatoryand antibacterial activity [112]. The selection of the polymers is basedon their ability to adhere to the mucosa and, simultaneously, retaintheir forms without fast degradation or swelling at acidic pH [112].The designed nanoparticle revealed antiadhesion and in vitro anti-

lin drug loaded.

182 D. Lopes et al. / Journal of Controlled Release 189 (2014) 169186H. pylori efcacy, with a MIC similar to that of metronidazole, neverthe-less it was signicantly higher than clarithromycin or amoxicillin [112].Compared to that observed with the unencapsulated GME and to freeclarithromycin, administration of GME-loaded nanoparticles during 3consecutive days revealed higher, although uncompleted, in vivo eradi-cation [112]. Recently (2014), Ali et al. used mucoadhesive micro-spheres to encapsulate curcumin, which is able to reduce theenhancement of metalloproteinases levels caused by H. pylori and re-sponsible for gastric injuries [113]. Mathematical models were used tooptimize the formulation and experimental results revealed that en-trapment efciency and in vitro bioadhesiveness increased with the in-crease of polymer concentration [113]. Release studies showed asustained release during 8 h [113].

Antacids are frequently used to complement antimicrobial therapyand SPs may be used to encapsulate them (Table 5). Pantoprazole is acommon antacid used to complement the therapy against H. pylori[114]. However, when it reactswith acid before absorption, its effective-ness signicantly decreases [114]. Thus, Rafn et al. (2006) usedmicro-particles composed of EudragitS100 andHPMC to encapsulate sodiumpantoprazole [114]. The aim of this researchworkwas the evaluation ofthe success of a novel preparationmethod, namely spray drying on pilotscale [114]. Validation studies ofmicroparticle preparation by pilot scaleshowed reproducible physicochemical properties as well as gastro-resistance prole [114].

A mixture of polymers has also been tested in order to produce vac-cines through the encapsulation of antigens in order to induce immuni-zation against H. pylori (Table 5). For instance, in 1999, a copolymercomposed of lactic acid and glycolic acid was used in order to achieveoral immunization [115]. In fact, poly(D-L-lactide-co-glycolide)(PLGA), a FDA approved biodegradable copolymer, was used to encap-sulate H. pylori lysates since it can be uptaken by M cells and carriedto the mesenteric lymph nodes and spleen [115]. Thus, it is possible touse these microparticles not only to protect the antigen but also to tar-get induction sites [115]. Results revealed that H. pylori lysate-loadedmicroparticles were able to induce immune system, namely Th2-typeresponses in Balb/c mice [115]. However this response was weakerthan that observed with cholera toxin-H. pylori immunized mice[115]. Further studies are necessary to optimize immunization sched-ules, doses and intervals [115]. H. pylori lysates were also incorporatedin a microsphere composed of poly(D,L-lactide)-polyethylene glycol co-polymer [116]. Due to their small size, microspheres could induce mu-cosal and systemic immune responses and, in fact, encapsulatedantigens achieved higher enhancement of specic antibodies than solu-ble antigen [116]. In 2012, Figueiredo et al. applied two based chitosannanoparticles, namely chitosan/sodium deoxycholate and chitosan/so-dium alginate/sodium tripolyphosphate, to induce immunizationagainst H. pylori [117]. Two different approaches were tested to con-struct a multiantigenic vaccine, specically DNA-vaccine and protein-vaccine, administered in two different routes (oral and intramuscular(i.m.)) [117]. Mucosal immunity was facilitated when chitosan-basednanoparticles were used through oral immunization, however i.m. im-munization achieved a more equilibrated cellular/humoral immune re-sponse [117].

3.3. Hybrid systems with liposomes and polymeric particles

Liposomes have been used as drug delivery systems due to the sev-eral advantages mentioned above. However, they can be unstable dur-ing storage and even in biological uids [28]. One of the major reasonsof their instability is the propensity to fuse with other liposomes, lead-ing to a payload loss [118]. Their instability is enhanced when in thepresence of acidic medium, divalent cations and blood cells [3,118].The coating with PEG is an alternative to overcome this limitation.Nonetheless, it is unsuitable to be used in the design of nanoparticlesfor the treatment of H. pylori because it makes fusion with the bacterial

membrane difcult by which drug release would occur [118].Furthermore, the loading capacity of liposomes is limited [119]. On theother hand, polymeric nanoparticles have mechanical stability withouthaving the valuable surface properties of liposomes [3]. Newhybrid sys-tems combining the advantages of liposomes and polymeric nanoparti-cles have been studied.

3.3.1. Polymeric core coated with a phospholipid bilayerHybrids composed of a polymeric core encased in a phospholipid bi-