A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery

Abstract

The pH-sensitive liposomes have been extensively used as an alternative to conventional liposomes in effective intracellular delivery of therapeutics/antigen/DNA/diagnostics to various compartments of the target cell. Such liposomes are destabilized under acidic conditions of the endocytotic pathway as they usually contain pH-sensitive lipid components. Therefore, the encapsulated content is delivered into the intracellular bio-environment through destabilization or its fusion with the endosomal membrane. The therapeutic efficacy of pH-sensitive liposomes enables them as biomaterial with commercial utility especially in cancer treatment. In addition, targeting ligands including antibodies can be anchored on the surface of pH-sensitive liposomes to target specific cell surface receptors/antigen present on tumor cells. These vesicles have also been widely explored for antigen delivery and serve as immunological adjuvant to enhance the immune response to antigens. The present review deals with recent research updates on application of pH-sensitive liposomes in chemotherapy/diagnostics/antigen/gene delivery etc.

• Chemotherapy
• drug/gene delivery
• fusogenicity
• intracellular delivery
• pH-sensitive lipid

Introduction

Liposomes are pharmaceutical carriers of choice for numerous practical applications such as for controlled and targeted delivery of drugs/antigen/DNA and/or diagnostic materials. The surface modified targeted liposomes serve as one of the smartest cargo capable of bypassing barriers imposed by the biological environment even up to cellular and sub-cellular levels (Paliwal et al., 2011). The liposomal products like Doxil® and Ambisomes® have reached the market and are being continuously used by clinicians. The development and application of novel targeting and/or release triggering schemes to improve the therapeutic index of drugs encapsulated within liposomes are widely reported in the literature. The composition of liposomal systems can be easily modified to facilitate triggered release in response to environmental conditions. For example, liposomes are specifically designed to control the release of their contents in response to acidic pH of the endosomal system. Such plasma stable liposomes are known as pH-sensitive liposomes (Hong et al., 2002). The concept of pH-sensitive liposomes tremendously improves the intracellular delivery of various materials such as anti-tumor drugs, toxins, proteins and DNA (Gerasimov et al., 1999; Roux et al., 2004). The pH-sensitive liposomes offer the protective effects of other liposomal formulations with specific environment-controlled drug release. Mechanistically, the pH-sensitive liposomes undergo rapid destabilization in acidic environments such as endosomes. They are generally composed of a neutral cone-shaped lipid dioleoylphosphatidyl-ethanolamine (DOPE) and a weakly acidic amphiphile, such as cholesteryl hemisuccinate (CHEMS) (Lai et al., 1985). The fusogenic behavior of these liposomes is due to the DOPE present in its lipid layer, which does not form bilayer structure when dispersed in aqueous media but form hexagonal structure. Another lipid such as dioleoylphosphatidylcholine (DOPC) (Webb et al., 1995) or N-succinyl-DOPE (Nayar & Schroit, 1985) can also be incorporated to induce pH sensitivity. These lipids have negatively charged group, which can be neutralized on acidification in endosome, leading to destabilization, fusion with endosomal membrane and content release (Drummond et al., 2000; Lutwyche et al., 1998; Venugopalan et al., 2002).

The pH-sensitive liposomes rapidly get destabilized in the acidic pH environment of the tumor tissue. However, the applications of pH-sensitive liposomes become limited due to their recognition and sequestration by the phagocytes of the reticulo-endothelial system (RES). This leads to a condition of very short circulation half-life of these carriers. To avoid their RES uptake and prolong circulation time, grafting of the liposomal membranes with pegylated phospholipids (PEG-lipids) is recommended (Momekova et al., 2010). In addition, the development of nano-size liposomes (about 150 nm or less) can contribute to the increase of the additional circulation time. The present review updates with recent development, mechanism and application of pH-sensitive liposomes as smart delivery vehicle for a variety of therapeutics.

The pH-sensitive liposomes in drug delivery

Liposomes offer numerous advantages as drug delivery systems, such as their size can be easily optimized for crossing the fenestration into interstitial space and efficient cellular uptake via endocytosis/phagocytosis. Furthermore, their surface can be equipped with a hydrophilic polymer, such as with a layer of PEG, to minimize the recognition and subsequent uptake by RES, and thus to have a longer circulation time for passive accumulation in cancer tissues via the enhanced permeation and retention (EPR) effect (Andersen et al., 2005; Yu et al., 2010). The EPR effect is schematically presented in Figure 1. Further, the availability of surface for easy modification using site-directing ligand provides opportunity for targeted liposomal drug delivery. The idea of pH-sensitive drug delivery was introduced in 1980 (Yatvin et al., 1980a,b). Later on, it was found that altered pH in different locations such as tumor extracellular environments is advantageous in the design of pH-responsive liposomes for specific cancer cell targeting, enhanced cellular internalization and rapid intracellular drug release (Bertrand et al., 2010; Sawant et al., 2006). Figure 2 depicts the mechanism of drug delivery via pH-sensitive liposomes. The intracellular delivery of cytotoxic drugs by the pH-sensitive liposomes presents an efficient means of overcoming the multidrug resistance, one of the causes for cancer treatment failures. Further, the anchoring of site-directing ligands on such pH-sensitive liposomes potentiates the effect by targeted drug delivery (Simard & Leroux, 2009, 2010).

Figure 1. Selective uptake of liposomes by enhanced permeation and retention (EPR) effect. The disrupted endothelial lining allows entry of liposomes to tumor tissues and absence of lymphatic drainage support the retention of drugs in the tumor area hence called enhanced permeation and retention effect (EPR).

Figure 2. Targeted drug delivery from pH-sensitive liposome. (A) Mechanism involved in intracellular drug delivery; [1] Destabilization of pH-sensitive liposomes triggers the pore formation in endosomal membrane, [2] Destabilization of liposomes release the encapsulated molecules in endosome which diffuse to the cytoplasm through the endosomal membrane, [3] Fusion between the liposome and the endosomal membranes. (B) Localized delivery of anticancer drug to tumor site.

The intracellular delivery of biologically active molecules using liposomes has been the subject of intensive research (Simoes et al., 2001, 2004). The pH-sensitive liposomes are able to interact and promote fusion or destabilization of endosomal membrane and efficiently release the encapsulated contents into the cell cytosol. The concept of pH-sensitive liposomes emerged from the fact that certain enveloped viruses developed strategies to take advantage of the acidification of the endosomal lumen to infect cells, as well as from the observation that some pathological tissues i.e. tumors, inflamed and infected areas, exhibit an acidic environment as compared to normal tissues. One of the mechanism triggering pH-sensitivity in liposomes is the combination of phosphatidylethanolamine (PE) or its derivatives with compounds containing an acidic group (e.g. carboxylic group) that act as a stabilizer at neutral pH. The pH-sensitive liposomes have been used to deliver anticancer drugs, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo (Slepushkin et al., 1997). Some of the recent reports are summarized in Table 1.

Table 1. List of recently published reports on pH-sensitive liposomes.

Drug	Delivery system	Concluding remarks	Reference
Doxorubicin	Multifunctional pH-sensitive immunoliposomes containing a pH-sensitive PEG-PE component, TATp, and the cancer cell-specific mAb 2C5	The nanocarrier demonstrated enhanced cytotoxicity and internalization by cancer cells.	Koren et al., 2012
Doxorubicin	Estrogen-anchored pH sensitive liposomes	The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. Serum enzyme activities such as LDH and CPK levels were also reduced suggesting reduced DOX induced cardiotoxicity.	Paliwal et al., 2012
Radioactive substance	PEG-folate-coated pH-sensitive liposomes	Animals treated with radioactive formulations showed a lower increase in tumor volume and a significantly higher percentage of necrosis compared with controls.	Soares et al., 2012
Deoxyribonucleic acid (DNA)	O-carboxymethyl-chitosan- cationic liposome-coated DNA/protamine/DNA complexes (CMCS-CLDPD)	In vitro and in vivo transfection studies confirmed that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS-CLDPD from serum interaction but also enhance gene transfection efficiency.	Li et al., 2012
Therapeutic peptide	pH sensitive stealth liposomes	Therapeutic peptide encapsulated in pH-sensitive stealth liposomes was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.	Ducat et al., 2011
Cisplatin	pH-sensitive liposomes	Stealth pH-sensitive-CDDP significantly reduced the renal toxicity.	Leite et al., 2009
Ovalbumin (OVA)	pH-sensitive fusogenic polymer-succinylated poly(glycidol)-(SucPG-) modified liposomes	The results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle. Immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 antibody response along with inducing Th1 and Th2 immune responses.	Watarai et al., 2013
Major histocompatibility complex (MHC) class I-restricted antigen	Cationic pH-sensitive liposomes	The exogenous protein antigen gained access to the class-I presentation pathway as the protein is introduced into the cytosolic compartment of the APC	Zhou et al., 1995
Man-LPR loaded with mRNA encoding a melanoma antigen	Mannosylated lipopolyplexes	The mRNA is formulated with histidylated liposomes and a histidylated polymer. These pH-sensitive liposomes promote membrane destabilization in endosomes upon the protonation of their histidine groups, allowed nucleic acid delivery in the cytosol. The DCs have been targeted via the mannose lipid incorporated in the liposomes.	Pichon et al., 2013
ISS-oligodeoxyribonucleotides (ODNs)	Listeriolysin O (LLO)-containing pH-sensitive liposomes	ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type	Andrews et al., 2012
HIV-1 V3 peptides	MPL adjuvanted pH-sensitive liposomes	liposomes composed of phosphatidylethanolamine-beta-oleoyl-gamma-palmitoyl (POPE)/cholesterol hemisuccinate (CHOH)/monophosphoryl lipid A (MPL) may be used as a potentially immunomodulating adjuvant system for the development of HIV and other viral vaccines.	Chang et al., 1999
Doxorubicin	Folate-poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine containing liposomes	A higher drug cumulative release from DOX-loaded liposomescontaining F-PEOz-DSPE or PEOz-DSPE in vitro was found in phosphate buffered saline at pH 5.0 medium than at pH 7.4. These results indicate that F-PEOz-DSPE exhibits selective targeting, pH-sensitivity and little cytotoxicity, and may be a promising polymeric material for dual receptor and pH-sensitive targeting liposome.	Xia et al., 2013
Temsirolimus	The amino acid-based lipids, 1,5-dioctadecyl-l-glutamyl 2-histidyl-hexahydrobenzoic acid (HHG2C(18)) and 1,5-distearyl N-(N-α-(4-mPEG2000) butanedione)-histidyl-l-glutamate (PEGHG2C(18)) containing liposomes	In vivo, CCI-779/PEGHG2C(18)-L after intravenous administration showed remarkably higher bioavailability and blood circulation time compared with unPEGylated CCI-779/HHG2C(18)-L, and had the strongest antitumor efficacy against xenograft renal tumor models.	Mo et al., 2013
pGPH1/GFP/Neo plasmid	Cationic liposomes composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE)	The complex of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes showed enhanced accumulation in tumor tissue in vivo compared with DC-Chol/DOPE liposomes alone.	Chen et al., 2013

Fattal et al. (2004) reported pH-sensitive liposomes for the “smart” delivery of antisense oligonucleotides. These are molecules that are able to inhibit gene expression, and potentially active for the treatment of viral infections cancer or inflammatory diseases. However, because of their poor stability and poor intracellular penetration, “smart” delivery systems such as anionic pH-sensitive liposomes are required. The liposomes composed of PE released their contents in response to acidic pH within the endosomal system while remaining stable in plasma, thus improving the cytoplasmic delivery of oligonucleotides after endocytosis (Fattal et al., 2004).

Chen et al. (2011) evaluated a novel modified pH-sensitive liposome with a cleavable double smart PEG-lipid derivative (mPEG-Hz-CHEMS) to clarify the mechanism. The mPEG-Hz-CHEMS is cleaved to pH-responsive hydrazone and esterase-catalyzed at ester linkages so that the PEGylated coating can be efficiently removed by the lower pH and esterase in the serum. This reduces or eliminates the accelerated blood clearance (ABC) phenomenon induced by repeated injection of PEGylated liposomes and hence pH-sensitive stealth liposomes have been proposed as improved carrier (Chen et al., 2011).

The pH-sensitive component

To understand the pH-sensitive behavior of liposomes, a study of the physiochemical aspects of pH-sensitive component is important. The phosphatidylethanolamine (PE), commonly used component in liposome, presents a minimally hydrated and small head group as compared to the respective hydrocarbon chains and exhibits a cone shape in spite of a lamellar phase (Seddon et al., 1983). The cone shape favors the formation of inverted hexagonal phase above the phase transition temperature due to interaction of amine and phosphate groups of the polar head group. When we intercalate amphiphilic molecules containing a protonatable acidic group in PE, it will form a bilayer structure stable at physiological pH (Lai et al., 1985). Although stable liposomes are formed at physiological pH, on acidification, protonation of the acidic groups of the amphiphiles leads to the destabilization of liposomes (Torchilin et al., 1993). Hence, selection of the pH-sensitive lipid and amphiphilic stabilizers is desired for the formation of stable pH-sensitive liposomes and determine the properties such as extent of cellular internalization, the fusogenic ability, pH sensitivity and stability in biological fluids. The dioleylphosphatidylamine (DOPE) is one of the key ingredients of pH-sensitive liposomes. The liposomes containing DOPE with phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC) and cholesteryl hemisuccinate (CHEMS), when incubated in acidic pH of endosomes, such liposomes undergo destabilization due to the presence of DOPE. This effect is mediated by low hydration of the polar head group of DOPE which is converted to a hexagonal inverted phase leading to the formation of non-lamellar structures and trigger destabilization at acidic pH (Litzinger & Huang, 1992). Table 2 lists some of the lipid/polymer components of pH-sensitive liposomes.

Table 2. Lipids or polymer used for the preparation of pH-sensitive liposomes.

S. No.	Lipid/polymer introduced for pH-sensitive character	Liposomal composition	Reference
1	PHC	Liposomes containing the pH-sensitive lipid PHC	Yatvin et al., 1980
2	pH-sensitive peptide GALA	pH-sensitive peptide GALA with DOTAP/DOPE	Subbarao et al., 1987
3	DOPE	pH-sensitive DOPE/CHEMS/PE–PEG liposomes	Woodle et al., 1992
4	Alkylated NIPAM copolymers	Alkylated NIPAM copolymers with EPC/Cholesterol liposomes	Zignani et al., 2000
5	POPE	POPE/CHOH/MPL	Chang et al., 2001
6	DDAB	EPC, DDAB, CHEMS and Tween-80	Shi et al., 2002
7	Diolein	Anionic pH-sensitive liposomes composed of diolein/CHEMS	Guo et al., 2002
8	SucPG	DPPC, DOPE, MPL, SucPG polymer	Watarai et al., 2013

DOPE: dioleoylphosphatidylethanolamine, EPC: egg phosphatidylcholine, CHEMS: Cholesteryl homosuccinate, DDAB: Dimethyldioctadecylammonium bromide, GALA: glutamic acid-alanine-leucine-alanine, NIPAM: Alkylated N-isopropylacrylamide, POPE: polyoleoylphosphatidylethanolamine, MPL: monophosphoryl lipid A, PHC: Palmitoyl homocysteine, SucPG: succinylated poly(glycidol), DPPC: Dipalmitoylphosphatidylcholine.

Novel pH-sensitive lipids

Some novel types of pH-sensitive lipids have been introduced for the formation of pH-sensitive liposomes. A liposome formulation consisting of egg PC and succinylated poly (glycidol), a PEG derivative having carboxyl groups is reported in literature. Although succinylated poly(glycidol) is not a pH-sensitive lipid itself; however a combination of this lipid with PC molecules results in the formation of a complex with geometry similar to that of DOPE. This phenomenon occurs due to the formation of hydrogen bonds between its hydroxyl group and an oxygen atom of the phosphate group of PC and hence under acidic conditions develops fusion ability with the endosomal membrane (Kono et al., 1994). Three different techniques have been used for the preparation of pH-sensitive liposomes in the absence of DOPE. First, the combination of cationic/anionic lipid is used for efficient vehicle for cytosolic delivery. The pH-sensitive liposomes containing EPC, dimethyldioctadecylammonium bromide (DDAB), CHEMS and Tween-80 undergo destabilization under acidic pH and showed retention of pH sensitivity in the presence of serum (Shi et al., 2002). Second, a lipid diolein with CHEMS in the ratio 6:4 have been reported for the preparation of anionic pH-sensitive liposomes. These liposomes were found to be stable at physiological pH, but rapidly released encapsulated content at pH 5.0 (Guo et al., 2002). The third formulation containing egg phosphatidylcholine (PC), cholesteryl hemisuccinate (CHEMS), oleyl alcohol (OAlc) and Tween-80 (T-80) as ingredients has been reported for its pH-sensitive characteristics in order to provide option to first generation pH-sensitive liposomes, based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE) (Sudimack et al., 2002). Results indicated that in comparison to DOPE-based pH-sensitive liposomes, the above formulations showed much better retention of their pH-sensitive properties in the presence of 10% serum. Additionally, such a system, when appended with ligands for folate receptor (FR) of tumor cells, showed approximately 17-times greater FR-dependent cytotoxicity in KB cells compared to FR-targeted non-pH-sensitive liposomes.

pH-sensitive polymers

The alkylated N isopropylacrylamide (NIPAM) co-polymer has been reported to confer pH sensitivity on liposomes. Incorporation of NIPAM to egg PC and cholesterol liposomes resulted in the formation of pH-sensitive liposomes that released the encapsulated moieties on reaching to the acidic environment (Leroux et al., 2001; Roux et al., 2004). Roux et al. (2002b) reported randomly alkylated NIPAM-anchored liposomes of ara-c. The developed liposomes showed a higher cytotoxicity towards J774 macrophage cell in comparison to liposomes without NIPAM. Various NIPAM-based copolymers have been synthesized and evaluated for the pH sensitivity, serum stability (Roux et al., 2002a, b, 2003) and stearic stabilization in sequential studies by a research group (Roux et al., 2002b, 2003).

Uptake and intracellular delivery of bioactive by pH-sensitive liposomes

The pH-sensitive liposomes are internalized more efficiently as phosphatidylehtanolamine (PE)-containing liposomes have the tendency of form aggregates, due to the poor hydration of its head group which can explain their high affinity to adhere to cell membranes (Seddon et al., 1983). The cone shape of PE molecules favors the establishment of strong intermolecular interactions between the amine and phosphate groups of the polar head groups, justifying the strong tendency of these molecules to acquire the inverted hexagonal phase above the phase transition temperature. While at physiological pH stable liposomes are formed, acidification triggers protonation of the carboxylic groups of the amphiphiles, reducing their stabilizing effect and thus leading to liposomal destabilization, since under these conditions PE molecules revert into their inverted hexagonal phase (Torchilin et al., 1993).

In case of ligand anchored pH-sensitive liposomes, the different steps are involved in the internalization and intracellular delivery (although numerous other approaches are also used for this purpose). After ligand binding to cell receptors, ligand anchored liposomes are internalized through the endocytotic pathway. The internalized liposomes will be retained in early endosomes, which mature into late endosomes. The fusogenic potential of pH-sensitive liposomes enables them for rapid destabilization at this stage, which prevents degradation of drug at the lysosomal level leading to their higher assessment to the cytosolic or nuclear targets. Interestingly, intracellular drug delivery via pH-sensitive liposomes is dependent on the pH drop in the endosome upon maturation to late endosome. This can be evaluated using lysosomotropic agents, which prevent endosome acidification. Overall, three mechanisms have been proposed for intracellular release of the drug from pH-sensitive liposomes. First, the destabilization of pH-sensitive liposomes triggers the destabilization of the endosomal membrane, most likely through pore formation, leading to cytoplasmic delivery of their contents (Collins, 1995). Second, upon liposome destabilization, the encapsulated molecules diffuse to the cytoplasm through the endosomal membrane. Third, the fusion between the liposome and the endosomal membranes leads to cytoplasmic delivery of their contents. The fusogenic properties of PE favor the hypotheses first and third.

Application of pH-sensitive liposomes

Table 3 lists the patents in the area of pH-sensitive liposomes. The various applications of these carrier systems are described in different sections as follows:

Table 3. List of patents on pH-sensitive liposomes.

S. No.	Patent No.	Title	Patent applicant
1	EP1438974 A2	pH-sensitive liposomes based on diacyl-glycero-phosphoethanolamine and diacyl-succinyl-glycerol	GE Healthcare As
2	EP0422543 A1	pH-sensitive liposomes	The Green Cross Coporation
3	US4925661A	Target-specific cytotoxic liposomes	Leaf Huang
4	US5786214A	pH-sensitive immunoliposomes and method of gene delivery to the mammalian central nervous system	Eric G Holmberg
5	US5965434A	Amphipathic pH sensitive compounds and delivery systems for delivering biologically active compounds	Vladimir Budker, Vladimir Gurevich, Jon A Wolff
6	US6426086B1	pH-sensitive, serum-stable liposomes	Demetrios Papahadjopoulos, Olivier Meyer, Jean-Christophe Leroux
7	US20040197392A1	pH-sensitive liposomes	Sigrid Fossheim, Knut-egil Loekling, Roald Skurtveit
8	WO2003032946A2	pH-sensitive liposomes for targeted drug delivery	Gertrude E Costin
9	2003/032946	pH-sensitive liposomes for targeted drug delivery	Petrescu Stefana, M Costin, Gertrude E Trif Mihaela, Nichita Norica, Dwek Raymond A
10	EP0983086	pH-sensitive liposomes and other types of encapsulated vaccines containing immunomodulators, and methods for making and using same	Jean-Claude Bystryn

The pH-sensitive liposomes in chemotherapy

The pH-sensitive liposomes have been prepared for numerous applications in drug delivery including chemotherapy (Karanth & Murthy, 2007). In a study, engineered drug carriers capable of spontaneous accumulation in tumor areas via the enhanced permeability and retention (EPR) effect have been reported. These liposomes were simultaneously bearing on their surface cell penetrating peptide such as TAT (transactivator of transcription) peptide (TATp) moieties and protective PEG chains. PEGylated liposomes are expected to accumulate in targets via the EPR effect, but inside the “acidified” tumor ischemic tissues lose their PEG coating due to the lowered pH-induced hydrolysis and penetrate inside cells via the now-exposed TATp moieties (Kale & Torchilin, 2007). Cisplatin is one of the most active cytotoxic agents and has been widely used in the treatment of carcinomas. A biodistribution study of stealth pH-sensitive liposomes containing cisplatin and free cisplatin, in solid Ehrlich tumor-bearing mice has been reported (Junior et al., 2007). After administering a 6 mg/kg single intravenous bolus injection of either free radiolabeled cisplatin or stealth pH sensitive liposomes containing radiolabeled cisplatin, blood and tissues were analyzed. The area under the concentration-time curve (AUC) obtained for blood using stealth pH sensitive liposomes administration was found to be 2.1 fold larger when compared with free cisplatin treatment (Araujo et al., 2011).

A similar report is also published very recently describing the importance of pH-sensitive liposomes for improved biodistribution and targeted delivery of cisplatin to tumor-bearing mice. Though long-circulating and pH-sensitive liposomes, containing cisplatin successfully avoid severe side effects as well as drug resistance and have been proven effective too. However, physical (i.e. aggregation/fusion) and chemical instabilities limit the use of these drug carriers as pharmaceutical products. Therefore, it is suggested that freeze-dried liposomes may be a successful strategy implemented to improve the stability of these formulations (dos Santos et al., 2011). Koren et al. (2012) reported pH-sensitive pegylated long-circulating liposomes modified with cell-penetrating TAT peptide (TATp) moieties and cancer-specific mAb. A degradable pH-sensitive hydrazone bond between a long shielding PEG chains and PE was introduced. The approach was using TATp conjugation with a short PEG₁₀₀₀-PE spacer and mAb to a long PEG chain (PEG₃₄₀₀-PE). The “shielding” effect of TATp by long PEG chains was the aim which was further investigated using three liposomal models. At normal pH, surface TATp moieties are “hidden” by the long PEG chains. Upon the exposure to lowered pH, this multifunctional carrier exposes TATp moieties after the degradation of the hydrazone bond and removal of the long PEG chains. Enhanced cellular uptake of the TATp-containing immunoliposomes loaded with doxorubicin was observed in vitro after pre-treatment at lowered pH. The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, this multifunctional immuno-Doxil® preparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. A multifunctional immunoliposomal nanocarrier was claimed for intracellular drug delivery after exposure to lowered pH environment, typical of solid tumors (Koren et al., 2012).

Jiang et al. (2012) designed dual-functional liposomes with pH-responsive cell-penetrating peptide and targeting ligand hyaluronic acid for tumor therapy. Paclitaxel-loaded hyaluronic acid (HA)-anchored pH-sensitive liposomes possess a remarkably stronger cytotoxicity toward the hepatic cancer (HepG2) cells at pH 6.4 as compared to at pH 7.4. The confocal laser scanning microscopy of coumarin 6-loaded HA-anchored liposomes showed efficient intracellular trafficking including endosomal/lysosomal escape and cytoplasmic release (Jiang et al., 2012). The engineered estrogen receptor (ER) targeted pH-sensitive liposomes for the site-specific intracellular delivery of doxorubicin (DOX) for breast cancer therapy have been developed by our group. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposome for drug targeting to ERs over-expressed by breast cancer cells. The estrone-anchored pH-sensitive liposomes (ES-pH-sensitive-SL) showed fusogenic potential at acidic pH (5.5). In vitro cytotoxicity studies, which were performed on MCF-7 cells, revealed that ES-pH-sensitive-SL formulation was more cytotoxic than non-pH-sensitive targeted liposomes (ES-SL). Further, in vivo biodistribution studies and antitumor activities of formulations were conducted on tumor bearing female Balb/c mice followed by intravenous administration. The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. The ES-pH-sensitive-SL accelerated the intracellular trafficking of encapsulated DOX, thus increasing the therapeutic efficacy. This suggested that estrone-anchored pH-sensitive liposomes may serve as one of the promising drug delivery carriers for the targeted intracellular delivery of anticancer agents to breast cancer with reduced systemic side effects (Paliwal et al., 2012). The PEG-coated pH-sensitive and PEG-folate-coated pH-sensitive liposomes containing the Gd-DTPA-BMA and radiolabeled have been reported to study the in vivo anti-tumoral activity on mice bearing solid Ehrlich tumor by Soares et al., 2012. The study revealed that after 31 days of treatment, mice treated with formulations had a lesser increase in tumor volume and a significantly higher percentage of necrosis compared with controls. Furthermore, mice treated with radioactive formulations exhibited lower weight gain without significant hematological or biochemical changes, except for toxicity to hepatocytes (Soares et al., 2012).

Cationic liposomes facilitate the cellular uptake by electrostatic absorptive endocytosis due to negative charge on the cell membrane (Bareford et al., 2007). These liposomes after internalization fuse with the endosomal membrane to release their contents into the cytoplasm (Koynova et al., 2006) or exhibit the proton sponge effect leading to swelling and subsequent disruption of endosomal membrane resulting into cytoplasmic delivery of intact liposomes (Shigeta et al., 2007). Although these liposomes posses capability of intracellular delivery, they are associated with the limitations such as cytotoxicity and rapid clearance from RES (Soenen et al., 2009). The incorporation of poy(ethylene glycol) modified lipid (PEG-lipid) into the membrane of cationic liposomes increase half life of liposomes to some extent, however, it impaired the interaction between liposomes and cell membrane of tumor cell (Moore et al., 2008). To circumvent this pH-sensitive hydrazone bond is incorporated between PEG and lipid that can be degrade in the tumor bioenvironment and shedding PEG for better interaction of carrier and cell membrane (Rodrigues et al., 1999). However, little success is achieved as the linkage cannot be broken in the extracellular environment (pH 6.0–7.0) but generally in the endosomal compartment (pH 4.0–6.0).

To exploit the merits of anionic liposomes (lower hematotoxicity), pegylated liposomes (longer circulation time), and cationic liposomes (enhanced uptake) Mo et al. (2013) developed pH-sensitive zwitterionic oligopeptide liposomes for intracellular delivery of temsirolimus (CCI-779). They developed and evaluated pH-sensitive liposomes based on zwitterionic oligopeptide lipids (HHG2C(18)-L and PEGHG2C(18)-L) as anticancer drug carriers for intracellular delivery and enhanced antitumor activity. The (HHG2C(18)-L; 1,5-dioctadecyl-l-glutamyl 2-histidyl-hexahydrobenzoic acid and PEGHG2C(18)-L); 5-distearyl N-(N-α-(4-mPEG2000) butanedione)-histidyl-l-glutamate are amino based lipid which have multistage pH-response to tumor bioenvironmental pH (pH 6.0–7.0) and endosomal/lysosomal pH (pH 4.0–6.0) successively. To understand localization of these carriers within the cell, coumarin 6-loaded pH-sensitive liposomes with these lipids were also prepared which showed higher tumor cellular uptake due to electrostatic absorptive endocytosis at (pH 6.5). This produced proton sponge effect for endo-lysosomal escape, and accumulated to the mitochondria based on stronger positive charge by the hydrolysis of a pH-sensitive linker at pH 5.5 and pH 4.5 (Mo et al., 2013).

The pH-sensitive immunoliposomes

Liposomal drug accumulation in the target tissue can be improved by attachment of the ligand capable of recognizing and binding to receptor present on the cell of interest. Antibody and their fragments are the most widely explored targeting moieties which can be easily anchored to liposomal surface without alteration of liposome integrity and the antibody properties (Torchilin, 1985). Immunoliposomes directed by monoclonal antibodies are promising vehicles for tumor targeted drug delivery (Abra et al., 2002; Kim et al., 2008). To further enhance the efficacy and cytosolic release of encapsulated content pH-sensitive immunoliposomes have been developed which are taken up by the target cell by ligand mediated endocytosis and upon internalization they fuses with the endosomal membrane at low pH.

Several laboratories exploited pH-sensitive liposomes for the delivery of therapeutic macromolecule (Connor et al., 1984; Duzgunes et al., 1985). The pH-sensitive liposomes containing herpes simplex virus thymidine kinase gene released content into the cytoplasm and delivered DNA expressed efficiently in the cells (Wang & Huang, 1986). To improve binding efficiency, target cells can be improved by using immunoliposomes (Huang et al., 1982). Wang & Huang (1987) reported immunoliposomes that bind to major histocompatibility antigen H2-Kk for the delivery of model gene Escherichia coli chloramphenicol acetyltransferase (CAT). Conner & Huang (1986) developed pH-sensitive immunoliposomes composed of dioleoylphosphatidylethanolamine and oleic acid containing cytotoxic drug 1-0-D-arabinofuranosylcytosine (ara-C) modified with anti-H-2Kk antibody. It was claimed that these pH-sensitive immunoliposomes efficiently delivered drug to target L-929 cells; however they were ineffective against nontarget A-3I cells (Conner & Huang, 1986).

Kim et al. (2009) developed long-circulating pH-sensitive liposomes with epidermal growth factor receptor antibody and were tested on A549 cells and BALB/c-nu/nu mouse tumor model. The developed formulations provided an efficient and targeted delivery of gemcitabine for tumors which over-express the EGFR (Kim et al., 2009). The knowledge of internalization pathway plays a fundamental role in optimizing drug targeting.

The pH-sensitive liposomes in tumor diagnosis

Liposomes are excellent candidates for the development of theragonostic agents and multimodal imaging probes. They release the entrapped imaging probe/radioactive agent in response to a change of physico-chemical variables like pH, redox potential, or concentration of specific enzymes that usually occur in the early asymptomatic stage of several diseases such as tumor. The pH-sensitive liposomes have also been utilized for diagnosis of tumor. The pH-sensitive liposomes entrapping 99mTc has been reported for scintigraphic imaging in Ehrlich tumor-bearing mice. The system showed high accumulation in tumor tissue with high tumor-to-muscle ratio and proposed as a potential agent for tumor diagnosis (de Barros et al., 2011).

In another report, paramagnetic pH-sensitive liposomes have been developed as imaging tools by magnetic resonance imaging (MRI). The proposed formulation allowed the fast and full release of Gadoteridol at pH 5.5. The leakage of the imaging reporter from the vesicles was associated with a relativity enhancement that allowed its visualization by MRI. It was claimed that the release mechanism implies the protonation of the basic sites that leads to vesicle aggregation, thus enabling the expression of the fusogenic property (Torres et al., 2011). Ferreira et al. (2012) developed long-circulating and pH-sensitive liposomes containing a 99mTc-labeled antibiotic, ceftizoxime, SpHL-(99mTc-CF) to identify osteomyelitis, an infectious disease located in the bone or bone marrow. The biodistribution studies showed a high values in the target-non target ratio and indicated that SpHL-99mTc-CF as a potential agent for the diagnosis of bone infections (Ferreira et al., 2012).

The pH-sensitive liposomes in vaccine delivery

The pH-sensitive liposomes are being explored as promising vehicles for delivery of therapeutic and prophylactic vaccines (Chang et al., 2001; Watabe et al., 2001). These liposomes are employed as effective means for delivery of small sized peptides, producing efficient immune response and reducing their toxicity (Tachibana et al., 1998). It is well known that efficient vaccination strategies have been required to overcome new upcoming pathogens. Dendritic cells are known as potent professional antigen presenting cells, they play a crucial role in innate and adaptive immune responses. The DCs recognize, take up, process and present antigens to native and resting T cells for induction of an antigen-specific immune response. One of the most effective strategies for efficient introduction of antigenic proteins into cytosol of DC is to use membrane fusion especially for membrane-based liposomes (Yoshikawa et al., 2008). Chang et al. (1999) developed V3 peptides encapsulated pH-sensitive liposomes to elicit the humoral and cell specific responses. No immune response was observed with the immunogen without liposomes, while antibodies and CTL response were detected with peptide encapsulated pH-sensitive liposomes. The results of this study conclude that immunogen encapsulated pH-sensitive liposomes may be used as a potentially immunomodulating adjuvant system for the development of HIV and other viral vaccines (Chang et al., 1999). In their another report, Chang et al. (2001) delivered cytotoxic T lymphocyte (CTL) epitopes successfully to target cells by encapsulation into pH-sensitive liposomes. They reported that CTL epitopes are transferred to endoplasmic reticulum (RE) to associate with class I major histocompatibility complex (MHC) molecules (Chang et al., 2001). Lee et al. (2002) investigated the immunization potential of pH-sensitive liposomes encapsulating fluorescein isothiocyanate (FITC)-conjugated H-2Kb CTL epitope. After 3 days of immunization, pH-sensitive liposomes elicited significant CTL activity against the delivered antigen. Authors suggested that pH-sensitive liposomes served as a strong peptide adjuvant, which may be useful for peptide delivery for the development of therapeutic or prophylactic vaccines (Lee et al., 2002).

Sakaguchi et al. (2008b) developed poly(glycidol) derivatives modified pH-sensitive liposomes. These polymer-modified liposomes delivered calcein into cytosol of HeLa cells after internalization via endocytosis and subsequent fusion with membrane of endosome or lysosome (Sakaguchi et al., 2008b). In this sequence Yuba et al. (2010) reported pH-sensitive fusogenic liposomes modified with carboxylated poly(glycidol) derivatives, such as polymer-succinylated poly(glycidol) (SucPG) and 3-methylglutarylated poly(glycidol) (MGluPG) that can deliver antigenic proteins ovalbumin (OVA) into the cytosol of DCs. The nasal administration of these liposomes activated the antigen-specific cellular immunity. The results suggested that the administration of the polymer-modified, OVA-loaded liposomes induced stronger cellular immune responses than the OVA-loaded plain liposomes. The MGluPG modified fusogenic liposomes exhibited high ability for CTL activation than SucPG. Thus it was concluded that MGluPG liposomes and their relevant liposomes might be promising antigen carriers for establishment of cancer immunotherapy and mucosal vaccination (Yuba et al., 2013). The pH-sensitive fusogenic SucPG modified liposomes as a vaccine carrier for ovalbumin (OVA) have been evaluated. After immunization only OVA-specific IgG1 antibody responses were observed with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes leads to the induction of OVA-specific IgG1, IgG2a and IgG3 Ab responses. The results suggested that the pH-sensitive SucPG modified liposomes may serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses (Watarai et al., 2013). Recently, Yuba et al. (2013) developed polymer modified pH-sensitive liposomes for the delivery of OVA with 3-methylglutarylated poly(glycidol) of linear or hyperbranched structure. These polymer-modified liposomes have been efficiently taken up by murine dendritic cells and subcutaneous or nasal administration of liposomes induced OVA-specific cytotoxic T cells much more effectively than the unmodified liposomes. These polymer-modified liposomes have been proposed as promising carriers of antigen for efficient cancer immunotherapy (Yuba et al., 2013).

The pH-sensitive liposomes in gene delivery

The increase in our molecular understanding, the number of biotechnological products such as nucleic acids, proteins and peptides are increasing as therapeutics for several clinical situations. However, the in vivo efficacy of these products can be severely compromised by their unfavorable physicochemical characteristics. The major obstacle in their usefulness is large size and hydrophilic nature. Additionally, macromolecules are often delivered to their target located in the cytoplasm; such molecules may not reach their target without a delivery system to overcome cellular barrier and facilitating cytosolic delivery (Leserman et al., 1990). Therefore, many different drug delivery systems including liposomes have been explored for this purpose. Liposomes are able to provide protection and targeting of the encapsulated macromolecule and may facilitate cellular internalization (Di Marzio et al., 2008; Pedroso de Lima et al., 2001). The success of gene therapy required the development of multifunctional carrier that could overcome various barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. A new gene delivery system was evaluated which consists of both the pre-condensed plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), and the blood protein transferrin (Tf) into the pH-sensitive liposomes (Rosa et al., 2008). They found that the transfection capacity of these systems was higher due to presence of ALA to pH-sensitive liposomes.

The mannosylated pH-sensitive lipoplexes containing cholesterol derivative, Man-His-C4-Chol, which possesses histidine (His) residues was reported for transfection (Nakamura et al., 2009). These lipoplexes showed higher cellular uptake suggesting that they are taken up by macrophages via mannose receptor-mediated endocytosis. The transfection efficiency was also higher in case of pH-sensitive derivative as compared to normal one. Khalil et al. (2011) reported a multifunctional envelope-type nanocarrier modified with octaarginine peptide (R8) and glutamic acid-alanine-leucine-alanine (GALA), as a pH-sensitive fusogenic peptide (R8-GALA-MEND) for liver gene delivery. The modification leads to higher gene expression efficiencies per nucleus-delivered pDNA. It was concluded that combination of a negatively charged core system and GALA modification of the R8-MEND could be efficiently used for delivering genes to the liver (Khalil et al., 2011). Macrophages express mannose receptors that can be exploited for targeted gene delivery to macrophages for the treatment of various immune diseases. Ducat et al., 2011 reported pH-sensitive stealth liposomes as a suitable vector for a therapeutic peptide (Print 3G, a peptidic agent that could reduce the angiogenic development of breast tumors) to its nuclear site of action (Ducat et al., 2011). They investigated cellular uptake of peptide-loaded liposomes in three cell lines; Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was observed as function of time. Using pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non-tumorigenic breast cancer cells (Ducat et al., 2011). These reports clearly demonstrate utility of pH sensitive liposomes as intracellular carrier for bioactive(s).

A pH-sensitive multifunctional gene carrying delivery system has been proposed that offered long circulation time but avoid the inhibition of tumor cellular uptake of carriers associated with the use of polyethylene glycol (Li et al., 2012). Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line and in vivo evaluation into tumor-bearing mice. The isolated tumor cells confirmed transfection efficiency by fluorescence determination (Li et al., 2012). CMCS-cationic liposome-coated DNA/protamine/DNA complexes had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. Similar other reports also describe importance of pH sensitivity within liposomal membrane for intracellular gene transfection (Sakaguchi et al., 2008a,b,c).

Chen et al. (2012) developed pH-sensitive lipoplexes for efficient gene delivery with the three novel cationic lipids containing a linear ortho ester linker that conjugates either the head group (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids bear counter ion either an iodide or a chloride. These lipids contain a trimethyl ammonium head group, two unsaturated oleyl hydrocarbon chains and a linear ortho ester linker. The Type I lipoplexes aggregated and destabilized at the endosomal pH (pH 5.5), but the Type II lipoplexes were not. The Type I ortho ester-based lipoplexes containing helper lipids, DOPE or cholesterol and with the chloride counter ion, significantly improved the gene transfection efficiency, more than 100 fold in CV-1 cells (monkey kidney fibroblast) (Chen et al., 2012).

Conclusion

The pH-sensitive liposomes have been explored as therapeutic carrier in different areas of drug delivery including imaging. The unique characteristics of these nanocarriers enable them as one of the preferred choices for formulation development in cancer therapy. The efficient intracellular delivery of bioactive(s) is highly desired to overcome multidrug resistance in case of chemotherapy, which can be achieved by the use of pH-sensitive liposomes. Additionally, components used for designing pH-sensitive liposomes do not impose any regulatory restrictions as they possess no toxicity when used in vivo. Since pH-sensitive liposomes are the modified version of existing and well accepted liposomes hence after proven pre-clinical and clinical effectiveness their commercial viability has prominent chances.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.