pH-responsive vaccine delivery systems for improving cellular immunity

Zonghua Liu,Linghong Huang,Wei Xue

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes,Department of Biomedical Engineering,Jinan University,Guangzhou,510632,China

Keywords:Vaccination Vaccine delivery systems Cellular immunity pH-responsive

ABSTRACT Immune vaccination is a critically important strategy in disease prevention and treatment.In vaccination,efficient vaccine carriers are necessary to augment the immune response initiated by vaccines generally with weak immunogenecity.Nowadays,commercially available vaccine/carrier formulations induce effective humoral immunity but weak or none cellular immunity.However,in practice,cellular rather than humoral immunity plays the key role in treating some intractable diseases such as AIDS and cancers,in which the elicited cytotoxic T lymphocytes can directly kill HIV-infected or cancer cells.To trigger potent cellular immunity,the carriers should help antigens escape from endosomal/lysosomal degradation and release them directly into the cytosol of antigen presenting cells.To this aim,such kind of vaccine carriers should be rationally designed and prepared in terms of their structure and physiochemical properties.Here,we summarized the recent advances in pH-responsive vaccine carriers exclusively developed for improving cellular immunity.

1.Introduction

Immune vaccination is an important strategy in disease prevention and treatment.Potent immune response induced by vaccine immunization requires the delivery of a sufficient amount of antigens to antigen presenting cells(APCs),which process and present the antigens to T lymphocytes.This involves a series of cellular processes including cellular binding to APCs as the first step,subsequent internalization,transfer from endosome into lysosome (or in some cases transfer from endosome into cytosol),complexation with major histocompatibility complex (MHC) molecules,and presentation on APC surface.For DNA vaccines,nuclear entry,gene transcription and antigen protein translation are extra necessary.For successful antigen delivery,proper carriers are indispensable at each stage.First,the immunogenecity of antigens is generally weak at the expense of satisfying safety requirement.Second,antigens are physicochemically labile to the harsh in vivo environment and generally are not able to enter cells by themselves,although they act intracellularly.To achieve desirable immunization efficacy,it is critical to rationally design delivery systems for antigens to efficiently pass through these cellular processes [1–11].

After captured by APCs,antigens are internalized and processed via two different pathways.The antigenic peptides originating from endogenous source are presented via MHC I molecules to native CD8+T lymphocytes,and the activated CD8+T lymphocytes further develop to cytotoxic T lymphocytes (CTLs) to elicit CTLs-mediated immune response(termed as cellular immunity).By contrast,exogenous antigensoriginated antigenic determinants are presented via MHC II molecules to target native CD4+T lymphocytes,and the activated CD4+T lymphocytes further differentiate and assist the antibody production of plasma cells to induce antibody-mediated response(termed as humoral immunity).CTLs can directly damage virus-infected cells or cancer cells,and hence cellular immunity is more effective than humoral immunity in the immune therapy of virus-infected diseases or cancers[12].

In actual immune vaccination,exogenous antigens deliberately administered are generally internalized through endocytosis,degraded in endosomes or lysosomes (and hence cannot reach the cytoplasm of APCs),and presented via MHC II pathway to induce humoral immunity.In some cases,however,exogenous antigens could also be presented via MHC I pathway to induce cellular immunity,which is termed as crosspresentation [13].The special cases of cross-presentation induced by exogenous antigens often require effective antigen endosomal and/or lysosomal escape and then antigen delivery into the cytoplasm of APCs,where the cytoplasmic antigen proteins are degraded into antigenic peptides and then assembled with MHC I molecules for further presentation.Orientation to cross-presentation of exogenous antigens and consequently to cellular immunity has important therapeutic ramifications for currently intractable diseases such as AIDS and cancers.Fortunately,proper antigen carriers can assist antigen escape from endosomal/lysosomal degradation and release the antigen directly into cytosol.In fact,endosomal/lysosomal escape and cytoplasmic delivery have gained tremendous attention in the design of intelligent carrier to deliver various drugs and biological(such as small interfering RNA and mRNA) [14–23].Therefore,a major challenge in inducing cellular immunity is to design antigen delivery systems that can efficiently orient antigen proteins to the cytoplasm of APCs to enhance MHC I antigen presentation.

To achieve efficient MHC I presentation and potent cellular immunity,various strategies have been attempted to design proper antigen carriers for cytoplasmic antigen delivery in APCs[24,25].Among them,pH-responsive antigen carriers have gained great attention.The pH-responsive antigen carriers can release exogenous antigen proteins into the cytosol of APCs,promote cross-presentation,and as a result boost cellular immunity.Here,we discussed the pH-sensitive antigen carriers exclusively developed to improve cellular immunity.For this aim,design strategies of such carriers include preparing pH-responsive polymer carriers by using acid-labile crosslinkers,pH-responsive polymer carriers by using acid-labile polymers,particularly pH-responsive poly(propylacrylic acid) (PPAA)-based polymer carriers,pHresponsive fusogenic liposomes,and pH-responsive carriers by using acid-sensitive membrane disruptors.Their detailed descriptions are specified at the beginning of each section,respectively.

2.pH-responsive polymer carriers by using acid-labile crosslinkers

In a primary trial,Frechet et al.found the hydrogels using the bisacrylamide acetal cross-linker (Fig.1A [26]) rapidly hydrolyze in acidic condition,with a half-life of 5.5 min at pH5.0,but 24 h at pH7.4.Within 2 h,along with the crosslinker hydrolysis,the FITC-bovine serum albumin loaded in the hydrogels was completely released at pH5.0,but only 5% at pH7.4.Such acid-liable crosslinker was further used to prepare acid-degradable microgels or nanogels for potential antigen delivery systems.

To achieve antigen endosomal/lysosomal escape and cytosolic delivery in APCs,acid-labile crosslinkers were used to prepare pH-responsive polymer carriers for antigen delivery by Frechet's group.Such crosslinkers could break in the acidic endosomal/lysosomal environment,causing the damage of the three-dimensional carrier network and then the following antigen release.A series of acid-labile crosslinkers(Fig.1A–E)were synthesized and used to copolymerize with acrylamide and/or other monomers by inverse emulsion/micro-emulsion polymerization to produce pH-sensitive hydrogel micro-or nano-particles.During the polymerization,the model antigen protein ovalbumin(OVA)was added in the aqueous phase to be physically embedded into the network of the particles.In this way OVA was not modified or denatured,and hence well maintained its natural immunogenicity as an antigen.The particles were relatively stable on neutral conditions.After the uptake of the particles by APCs,the acid-labile crosslinkers were cleaved by acid-catalyzed hydrolysis in the acidic environment of the endosomes of APCs,as displayed in Fig.1F.The rapid cleavage of the crosslinkers caused particle degradation,phagosomal disruption through a colloid osmotic mechanism,antigen escape from the endosomes and then antigen release into the cytoplasm,which promoted antigen cross-presentation and the enhancement of CTL response.The acid-labile crosslinkers-based micro or nano-gels found applications as vaccine carriers for improving cellular immunity to treat viruses-infected diseases and tumors.

In early studies,the group synthesized acid-labile benzyl acetal cross-linkers.First,by using an acid-liable benzyl crosslinker (Fig.1B[27]),OVA was loaded into acid-degradable microgels (spherical,200–500 nm in the dry state) synthesized by inverse emulsion polymerization with OVA in the aqueous phase.The encapsulated OVA amount was 9–11 μg per mg of microgels,reaching 50% encapsulation efficiency.In the LacZ assay,B3Z cells(a CD8+T cell hybridoma)were used to evaluate the antigen cross-presentation by MHC I molecules.The OVA loaded in the microgels was several orders of magnitude higher to elicit CTLs response,compared to free OVA.Other immune studies were not conducted here.The microgels up to 5 mg/mL displayed the minimal cytotoxicity.Then,the group fabricated a more hydrophilic acid-liable benzyl cross-linker(Fig.1C[28])by adding one more ethylene oxide unit than the above crosslinker.This enhanced water-dispersibility and OVA loading efficiency of the resulting particles (spherical,250–500 nm).The maximum encapsulation was up to 154.8 μg OVA/mg particles.A 2.5-fold increase of OVA loading resulted to a 20-fold up-regulation of antigen presentation in the LacZ assay.In an in vivo test,after two preventive subcutaneous vaccinations,EG7 tumor was established.The mice immunized with OVA-loading particles rejected EG7 tumor cells,and their survival rate was 100% after 17 d,compared with about 14% (free OVA group) and 7% (saline control).Later,they [29] extra introduced a cationic monomer,2-acryloxyethyltrimethylammonium chloride into the polymerization to prepare acid-degradable cationic nanoparticles (180–230 nm determined by dynamic light scattering,and 217 ± 49 nm by transmission electron microscopy) by using the same acid-labile benzyl acetal cross-linker (Fig.1C).The nanoparticles as carriers enhanced antigen cross-presentation in bone marrow derived dendritic cells(BMDCs) as quantified by the LacZ assay.The cationic particles promoted the antigen internalization by BMDCs.Additionally,adjuvants CpG and anti-interleukin-10 oligonucleotides could also be co-delivered with OVA by adsorbing onto the particles to maximize cellular immunity.

In the following studies,the group synthesized wholly aliphatic acid-labile acetal cross-linkers.They [30] newly synthesized an aliphatic monomer bearing a pendant primary amine and an acid-liable acetal bond,and a wholly aliphatic acid-cleavable acetal cross-linkers(Fig.1D) to prepare acid-cleavable cationic microparticles (4–5 μm determined by dynamic light scattering) to improve biocompatibility and to accelerate hydrolysis.Introduction of such a monomer into the particles increased the OVA loading efficiency up to 114 μg/mg particles,approximately 5–10 times higher than the previous method [27],owing to the electrostatic attraction of the cationic groups with OVA.Moreover,addition of 10% cationic acid-liable monomer into the particles augmented ca.75 times higher MHC I antigen presentation than that using non-degradable particles.In view of the low number of dendritic cells (DCs) in normal tissues,positive targeting DCs could enhance immune response.Hence,the OVA-loading pH-degradable cationic particle carriers was further conjugated with DCs-targeting anti-DEC-205 (anti-CD205) monoclonal antibodies [31].The IFN-γ-secreting CD8+T cells elicited by the vaccinated mice were 5% for immunization with the anti-DEC-205 antibody conjugated particles,2.5%for immunization with the isotype antibody-conjugated particles,and 0.6% for immunization with free OVA,respectively.

It is important to deliver antigen and adjuvant into the same APCs.To this aim,the immune adjuvant CpG was conjugated to a monomer,and the resulting CpG-bearing macromonomer further copolymerized with acrylamide and the acid-liable crosslinker (Fig.1C [32]) to prepare acid-sensitive CpG-displaying particles (spherical,250–500 nm in dry state).The antigen and adjuvant encapsulation was 40 μg OVA/mg particle and 25 μg CpG/mg particle,respectively.The conjugation of CpG to the particles increased 45-fold of interleukin-12 level and significantly up-regulated the expression of co-stimulatory molecules of APCs.Moreover,immunization with such CpG-displaying particles elicited stronger cellular immune response in vivo,including increased antigen-specific CD8+T cell proliferation,cytokine IFN-γ secretion level,and antigen-specific cytotoxicity.Furthermore,by adopting a 3′-conjugation method,the group optimized the covalent linkage of pG adjuvant to the carrier particles,and the loadings amount was up to 9 μg CpG/mg particles.The obtained particles (～100–800 nm in dry state) could simultaneously release protein antigen and CpG in acidic environment [33].From an in vivo cyto-toxicity assay,the co-delivery of OVA antigen and CpG by the same carrier contributed to stronger CTL activity than the particles only mixed with CpG.These particles produced effective immune protection against MO5 murine melanoma.Moreover,it was found that 3′-conjugated CpG particles were more immune-stimulatory than 5′-conjugated CpG particles.

Fig.1.Chemical structures of acid-labile crosslinkers:A [26],B [27],C [28,29,32–34],D [30,31],E [35],and their action mechanism F [34].

The group further investigated different physicochemical properties and surface modifications of the carrier particles on their delivery efficacy.First,they synthesized different sizes (3.5 μm and 35 nm) of OVA-loading particles and compared their effect of on immune responses [34].The in vivo evaluations indicated that the different sizes of particles were both effective at triggering immune responses,but had no significant difference in T-cell activation.Besides,they[35]utilized a novel acid-liable crosslinker (Fig.1 E) to prepare OVA-loading pHsensitive particles (1.5 μm) decorated with functional antibodies to explore if DCs-targeting antibody (anti-DEC205),hemagglutinin antibody (anti-HA,as a negative control) and/or activating antibody(agonistic anti-CD40) could affect the APCs-particles interactions.The results showed that,the particles were internalized by BMDCs in vitro and by both DCs and monocytes in vivo.The particles were also upregulated the co-stimulating molecules (CD80 and CD86) of BMDCs in vitro and DCs in vivo.Both the internalization by and activation of the APCs were not related to the conjugated antibodies.The particles also amplified the CD8+T cells against the infection of OVA-expressing vesicular stomatitis virus.

3.pH-responsive polymer carriers by using acid-labile polymers

Despite the unique advantage of the acid-labile crosslinkers-based polyacrylamide particles as a proof of concept system,there could be biosafety concern with the in vivo accumulation of the high molecular weight of nonbiodegradable polyacrylamide byproducts remaining after the crosslinker degradation.Biocompatibility issue necessitates the use of fully acid-degradable materials for cytosolic antigen delivery.The rationale for this design is based on a colloid osmotic mechanism,namely,a rapid decomposition of the carrier materials to many molecules causes endosome disruption by increasing the osmotic pressure and then a rapid water influx in the endosome.Rupture of the endosomal compartment leads to antigen leakage to the APC cytoplasm,circumventing the possible antigen degradation in the acidic endosome[28].

First,Frechet et al.[36]utilized a fully degradable polymer,acetalderivatized polyurethane (Fig.2A),to prepared acid-degradable particles as antigen carriers,which could degrade to low molecular weight of nontoxic byproducts,as shown in Fig.2A.The particles were fabricated with a double emulsion method and simultaneously encapsulated OVA by using OVA solution as the aqueous phase.The particles were 100–500 nm in diameter with an average of 249 ± 5 nm with the OVA encapsulation efficiency of 25%.When co-cultured with RAW macrophages,the degradation byproduct of the particles reached its peak level at 24 h,and was undetectable after 4 d.When tested in vitro,the particles elicited a great increase in the MHC I presentation in both macrophages and DCs.Immunization with the particles produced higher CTL response than those with free OVA or slower-degrading analogous particles.

Fig.2.Chemical structures of acid-responsive polymers:A,acetal-derivatized polyurethane [36];B,acetal-derivatized dextran [37].

Furthermore,Frechet et al.[37–40] prepared the acid-responsive biodegradable acetal-derivatized dextran for antigen delivery,as shown in Fig.2B.Transforming the hydroxyl groups of dextran to acetals made the “acetalated-dextran” hydrophobic and enhanced its solubility in organic solvents.Thus,the resulting hydrophobic material could be easily processed to nanoparticles using emulsion techniques.Moreover,acetalated dextran further displayed pH-sensitivity since the pendant acetal groups could hydrolyze under mildly acidic aqueous conditions,which regenerated native dextran with a low amount of small molecule byproducts.The acetalated dextran was assessed to have good biocompatibility and the resulting nanoparticles greatly increased MHC I presentation.Moreover,degradation rate of acetalated dextran microparticles could be adjusted by controlling the degree and type of acetal modification,further contributing to desirable antigen presentation[38].In addition,Ma et al.[41] designed amphiphilic pH-sensitive galactosyl dextran-retinal nanogels as tumor antigen carriers,by conjugating retinal to dextran with a pH-sensitive hydrazone bond,and then by galactosylation to target DCs.The encapsulation efficiency of OVA in the nanogels was over 80%,and the obtained nano-vaccines were spherical with hydrodynamic size of～100 nm and zeta potential of +16 mV.The pH-sensitive nanogels could promote DCs maturation by activating retinoic acid receptor signaling,and also promote antigen internalization and cytosolic delivery by DCs.The nanogels efficiently enhanced MHC I antigen presentation,and elicited strong anti-tumor responses in vivo.

Besides,conjugation of antigens to pH-labile polymer carriers is also a good strategy to deliver antigen in a pH-responsive manner.Kong et al.[42] crosslinked two types of functionalized alginates with CaCl2to fabricate an antigen carrier.A part of alginates was modified with mannose to target DCs.Another part of alginates was conjugated with OVA via pH-sensitive Schiffbase bond.The obtained spherical nanoparticles with hydrodynamic size of～310 nm and negative surface charge of -45.6 mV,could facilitate the antigen uptake and cytosolic release by BMDCs,the secretion levels of cytokines and the expression of the co-stimulating molecules of which also significantly up-regulated,compared with free OVA.In vitro,the nanoparticles could augment the cross-presentation of OVA in B3Z T cell hybridoma.Subcutaneous injection of the nanoparticles could elicit the CTL response of C57BL/6 mice and inhibit E.G7 tumor growth.In addition,Zhang et al.[43] used redox-sensitive disulfide bond to conjugate OVA to a pHsensitive carrier,amphiphilic poly(L-histidine)-poly(ethylene glycol)micelles,to prepare both redox-and pH-sensitive vaccines.The conjugation efficiency of OVA to the blank micelles via disulfide bond was 94.82% ± 1.89.The OVA-loading micellar vaccines were spherical or ellipsoidal with the size of 124.0 ± 4.78 nm and zeta potential of-9.98 ± 1.50 mV.After subcutaneous or intraperitoneal injection of the vaccines,the amounts of CD3+CD8+T cells,CD3+CD8+25D11.6+T cells,IFN-γ,and IL-2 increased,proving the enhancement of antitumor immune therapy of the vaccine formulation.It is worth noting that poly(L-histidine) has good biodegradation,biocompatibility and low toxicity.Moreover,the imidazole groups of poly(L-histidine)can be protonated in acid environment,leading to proton pump effect.Along with the protonation of imidazole rings,the solubility of poly(L-histidine) changes,hydrophobic (uncharged) at pH7.4 and hydrophilic(positively charged) at pH5.

Some pH-sensitive polycations that can degrade in acidic environment,are ideal antigen carrier materials,because they can adsorb antigens via electrostatic attraction and then cause proton sponge effect to realize endosomal/lysosomal escape of antigens.pH-Sensitive poly(βamino ester) is such a rare polycation.Tan et al.[44] synthesized pHsensitive mannose-modified poly(β-amino ester) to co-deliver tumorassociated antigen peptide Trp-2 and adjuvant monophosphoryl lipid A.By additional covalent introduction of 18.2% stearylamine to poly (βamino ester) chains,the encapsulation efficiency of the Trp-2 peptide reached up to 76.5%.The obtained spherical nano-vaccines with the mean hydrodynamic diameter of 147 nm and zeta potential of 11.0 ± 2.9 mV,could target and mature DCs,and hence improved the killing activity of antigen-specific CTL against melanoma.The vaccine was also co-administered with an immune checkpoint inhibitor (PD-L1 antagonist) to relieve the immunosuppression in tumor microenvironment.In a prophylactic study,the co-administration of the vaccine and the PD-L1 antagonist augmented the anti-tumor efficacy by 3.7 fold,and prolonged median survival time by 1.6-fold,compared than only immunization of the vaccine.

Biodegradable poly(orthoester)s have been a type of attractive medicament materials since 1970s.Moreover,poly(orthoester)-formulated antiemetic has recently been approved by FDA for clinical application.However,the degradation ratio of poly(orthoester) in acidic environment is not ideal and has been a hurdle for its extensive applications.To overcome this disadvantage,Guo et al.[45] synthesized poly(acyclic orthoester)s that can rapidly hydrolyze at lysosomal pH.At pH5,hydrolysis of the poly(acyclic orthoester)s nanoparticles(～250 nm)reached 80%in 2 h,and their half-lives varied from 0.31 to 0.43 h.OVA-loading poly(acyclic orthoester)s nanoparticles(100–150 nm by transmission electron microscopy) could efficiently deliver OVA into the cytosol of DCs,and enhanced antigen presentation compared with poly(lactic-co-glycolic acid) (PLGA) nanoparticles.

Fig.3.Chemical structures of PPAA-based polymers:A,poly(PAA-co-PDSA) [47];B,poly(BMA-co-DEAEMA)-b-poly(DMA-co-PDSMA) with different architectures[52];C,PPAA-b-poly(DMA-co-PDSMA) [49];D,poly(PDSEMA-co-DMAEMA)-b-poly(PAA-co-BMA-co-DMAEMA) [50];E,Poly(BMA-co-DMAEMA-co-PAA)-b-poly(HPMA-co-PDSMA) [51].

4.pH-responsive poly(propylacrylic acid) (PPAA)-based polymer carriers

PPAA mimics the pH-responsive endosomolytic activity of some viruses,the fusogenic coat proteins of which have a pH-triggered conformational transformation to an endosomolytic state and then make the viruses evade lysosomal degradation [46].At physiological pH,PPAA in the ionized state has not membrane-disruptive activity.However,the carboxyl groups of PPAA become protonated and the polymer transits to a membrane-destabilizing state by solubilizing liposomal lipid molecules in the weakly acidic solution (pH 5.5–6.5) of the endosomes and lysosomes of APCs.Due to the unique endosomolytic activity of PPAA,Stayton's group developed PPAA-based vaccine delivery systems that displayed pH-sensitive,membrane-destabilizing activity,as discussed below.

(1) Poly[PAA-co-pyridyldisulfide acrylate (PDSA)] abbreviated as poly(PAA-co-PDSA) [47] For antigen loading,OVA was conjugated to poly(PAA-co-PDSA)by disulfide exchange,as shown in Fig.3A.The reversible conjugate could deliver antigen into the cytoplasm of APCs by the reduction of the glutathione redox system.The poly(PAA-co-PDSA)-OVA conjugate contained 37% w/w of OVA and 63% w/w of the polymer poly(PAA-co-PDSA),or 0.588 μg OVA/μg polymer.The molecular weight measurement showed Mw=200 kD,Mn=45 kD,PDI=4.4 for the conjugate.The conjugate displayed the pH-responsive membrane-disruptive activity of the carrier polymer,and consequently elicited 22-fold increases in the MHC I display and OVA-specific CTL level compared with free OVA.

In a further study [48],OVA was conjugated via disulfide bonds to the carrier polymer poly(PAA-co-PDSA) (Mn=22 kD and Mw/Mn=2.0,with 3 mol%PDSA),forming a water-soluble OVA-poly(PAAco-PDSA) conjugate (Mn=43 kD and Mw/Mn=2.9) containing 38%w/w OVA.The conjugate was further condensed with a polycation poly(dimethylaminoethyl methacrylate) (PDMAEMA) (Mn=7.8 kD and Mw/Mn=1.2),to form OVA-poly(PAA-co-PDSA)/PDMAEMA particles with the hydrodynamic radius of 178 ± 45 nm and the zeta potential of-32 mV.In a prophylactic anti-EG.7-OVA tumor test,both the OVApoly(PAA-co-PDSA)conjugate and OVA-poly(PAA-co-PDSA)/PDMAEMA particles greatly suppressed tumor growth,and increased～3.5-fold in tumor-free survival longevity compared to controls.This was attributable to 8-fold up-regulation in OVA-specific CTL production and 11-fold up-regulation in anti-OVA IgG level.

(2) PPAA-b-poly[N,N-dimethylacrylamide (DMA)-co-(pyridyldisulfide methacrylamide) (PDSMA)] abbreviated as PPAA-b-poly(DMA-co-PDSMA) [49] In the above poly(PAA-co-PDSA) carrier polymers,pyridyldisulfide functionalized acrylate monomers were randomly copolymerized,potentially causing unexpected cross-linking and further disordered protein conjugation.To overcome the shortcoming,the group prepared the PPAA-b-poly(DMA-co-PDSMA) diblock copolymer with the absolute molecular weight of 2.8 kDa and PDI of 1.63,as shown in Fig.3C.The PPAA block as the endosomolytic component could freely act on endosome membrane,and the second short block with less pyridyldisulfide groups could decrease unfavorable cross-linking.The copolymer was further conjugated with OVA to form PPAA-b-poly(DMA-co-PDSMA)–OVA conjugate vaccines.In a prophylactic anti-EG.7-OVA tumor test,the mice vaccinated with the PPAA-b-poly(DMA-co-PDSMA)–OVA conjugate,free OVA,and PBS had a survival rate of 100%,40%,and 20% at day 21,respectively.

(3) Poly[pyridyldisulfide ethyl methacrylate (PDSEMA)-co-dimethylaminoethyl methacrylate (DMAEMA)]-b-poly[PAA-co-butylmethacrylate (BMA)-co-DMAEMA] abbreviated as poly(PDSEMA-co-DMAEMA)-b-poly(PAA-co-BMA-co-DMAEMA) [50] The PPAAbased,amphiphilic diblock copolymers (as shown in Fig.3D) assembled to pH-responsive,endosomolytic micelles (23 nm) with simultaneously conjugating antigen OVA and physically adsorbing adjuvant CpG.In the diblock copolymer,the ampholytic coreforming block was responsible for micelle formation and endosomal escape,and the polycationic shell block pendent with pyridyldisulfide groups was responsible for OVA conjugation and CpG complexation.Vaccination with the OVA-conjugated micelle/CpG formulation augmented CD8+T cell responses (3.4% IFN-γ+of CD8+) 7-,18-,and 8-fold compared to those with OVA-conjugated micelle,OVA/CpG mixture,and OVA/CpG/micelle mixture,respectively.The OVA-conjugated micelle/CpG formulation also significantly enhanced CD4+IFN-γ+(Th1) response and induced a balanced IgG1/IgG2c response.

(4) Poly(BMA-co-DMAEMA-co-PAA)-b-poly[N-(2-hydroxypropyl) methacrylamide (HPMA)-co-PDSMA] abbreviated as Poly(BMA-co-DMAEMA-co-PAA)-b-poly(HPMA-co-PDSMA) [51] The copolymer was composed of a hydrophilic HPMA shell with pendent pyridyldisulfide functionalities for conjugating OVA,and a pH-sensitive,endosomolytic,ampholytic core containing DMAEMA,PAA,and BMA,as shown in Fig.3E.The OVA-conjugated copolymer selfassembled into pH-responsive,endosomolytic,OVA-loading micelles(25–30 nm).In an in vitro test,DC2.4 cells(a cell line of DCs)co-cultured with the OVA-loading micelles exhibited 67-fold higher cytosolic OVA accumulation amount compared to free OVA at 4 h after internalization.The mice vaccinated with the OVA-loading micelles significantly increased OVA-specific CD8+T cell responses(0.4% IFN-γ+of CD8+) relative to those with free OVA,OVA/micelle mixture,and the control micelle without endosomal escape ability.

(5) Poly[BMA-co-2-(N,N-diethylamino)ethyl methacrylate(DEAEMA)]-b-poly(DMA-co-PDSMA) abbreviated as poly(BMA-co-DEAEMA)-bpoly(DMA-co-PDSMA) [52].The copolymer was prepared to construct pH-responsive,endosomolytic diblock copolymer nanoparticles with linear,core-crosslinked,or hyperbranched architectures,as shown in Fig.3B,to compare the effects of different carrier copolymer architectures on antigen delivery efficacy.The copolymer chains self-assembled into 25 nm micelles and simultaneously conjugating thiolated OVA.The copolymers displayed pHdependent membrane-disruptive activity,with the hyperbranched and crosslinked constructs showing significantly higher activity at pH ≤7.0 than the linear one.Also,the hyperbranched and crosslinked constructs augmented in vitro MHC I presentation compared to free OVA,while the linear one did not show a significant effect.The hyperbranched construct increased 4–5 fold of MHC I presentation compared to the crosslinked one.

5.pH-responsive fusogenic liposomes

Kono's group developed pH-responsive liposomes by modifying stable liposomes with pH-responding membrane-disruptive polymers,including pH-sensitive poly(glycidol) derivatives and pH-sensitive dextran derivatives,as discussed below and shown in Fig.4.Generally,liposomes are internalized by cells mainly via endocytosis,then transported by endosomes and finally degraded in lysosomes.The pHresponsive liposomes are membrane-friendly at neutral pH,but become membrane-disruptive by fusing with endosome membrane in the mildly acidic solution of endosomes,causing cargo leakage into the cytosol.It is assumed that,the destabilization of liposome membranes caused by the pH-sensitive fusogenic polymers may derive from the hydrophobic interaction and hydrogen bond formation between the polymer molecules with the liposome membrane [53].

(1) carboxylated liner poly(glycidol) (LPG) derivativesFirst,four carboxylated LPG derivatives with varying hydrophobicity (as shown in Fig.4B) were compared in terms of their respective abilities to endow the pH-sensitivity activity of egg yolk phosphatidylcholine liposomes [53].The derivatives were water-soluble at physiological pH but precipitated in mildly acidic pH.The derivatives with the more hydrophobic side chains precipitated at higher pH.The liposomes bearing higher hydrophobic derivatives exhibited more intense pH responses at weakly acidic pH and hence more efficient cytosolic delivery.By comparison,1,2-cyclohexanedicarboxylic anhydride modified LPG derivative destabilized liposomes even at neutral pH,while 3-methylglutaric anhydride modified LPG derivative produced highly potent pH-sensitive liposomes.

Among the four pH-responsive fusogenic liposomes,succinylated LPG-and 3-methylglutarylated LPG-modified,pH-responsive fusogenic liposomes were used for gene delivery to DC2.4 cells.It was shown that more pH-responsive liposomes achieved better transfection efficiency,implying that endosomal escape was critical for efficient transfection[54].The two pH-responsive fusogenic liposomes were further investigated as protein antigen carriers for improving cellular immunity[55].The pH-responsive liposomes were internalized efficiently by DC2.4 cells,then achieving cytosolic antigen release.The liposomes elicited cross-presentation in BMDCs.Nasal administration of the pHresponsive fusogenic liposomes induced stronger cellular immunity than plain liposomes.Besides,the two pH-responsive liposomes were also used for mucosal immunizations such as nasal,intraocular [56] or intraperitoneal routes [57].

In addition,the co-delivery potential of OVA and IFN-γ gene was tested for tumor-specific immunotherapy [58].The carriers were designed by using 3-methylglutarylated LPG-modified pH-sensitive liposomes for delivering OVA and cationic liposomes for delivering IFN-γencoding plasmid DNA,respectively.After mixing in aqueous solution,the OVA-loaded liposomes and the DNA-loaded cationic liposomes (lipoplexes) interacted via electrostatic attraction and formed hybrid complexes.Subcutaneous vaccination with the hybrid complexes decreased the tumor volumes of the mice carrying E.G7-OVA tumor.Subcutaneous injection of the liposomes and the lipoplexes triggered an extremely strong anti-tumor immune response.

(2) carboxylated hyperbranched poly(glycidol) (HPG) derivativespH-Sensitive carboxylated HPG derivatives (as shown in Fig.4A)were further prepared for liposome modification [59].Compared with the carboxylated LPG,the carboxylated HPG demonstrated a stronger interaction with liposome membrane and more effective cytosolic antigen delivery in DC2.4 cells.This shows that the architectural structure of poly(glycidol) played an important role in their pH-sensitivity and interaction with endosomal membranes.In subcutaneous or nasal vaccination of OVA,3-methylglutarylated LPG-or 3-methylglutarylated HPG-modified liposomes effectively induced CTLs [60].Furthermore,OVA delivery by the pH-responsive liposomes significantly decreased the tumor burden of the mice bearing E.G7-OVA-tumor.This suggests that the highly pHsensitive liposomes were promising vaccine carriers for efficient cancer treatment.

Furthermore,the same group [61] also used 3-methyl-glutarylated HPG-modified liposomes to deliver OVA antigenic peptides OVA-I(SIINFEKL)and OVA-II(PSISQAVHAAHAEINEAP-A),which bind to the MHC I and II molecules on DCs,respectively.The peptides could be delivered to the cytosol of DCs.The E.G7-OVA-tumor bearing mice immunized with OVA-I-loading 3-methyl-glutarylated-HPG-modified liposomes,could induce much more efficient OVA-specific CTL activation than free OVA-I peptide,and efficiently inhibited the tumor volume.Moreover,the group [62] incorporated the immune adjuvant CpG into the 3-methyl-glutarylated HPG-modified positive liposomes by using pre-mix and post-mix methods to further enhance antigenspecific immunity.Both methods more efficiently delivered CpG into the endosomes of DCs than free CpG,and induced more potent antitumor efficacy.

Fig.4.Chemical structures of pH-sensitive fusogenic polymers for liposome modification:A,carboxylated HPG and LPG derivatives [59];B,carboxylated LPG derivatives [53];C,3-methylglutarylated LPG-and 2-carboxycyclohexane-1-carboxylated LPG-phosphatidylethanolamine conjugates [64];D,3-methylglutarylated dextran derivatives [63].

(3) 3-methylglutarylated dextran derivativesTo overcome the nondegradability of poly(glycidol),Kono et al.further prepared 3-methylglutarylated dextran derivatives for surface modification of liposomes (as shown in Fig.4D) [63].The so-obtained liposomes were membrane-friendly at neutral conditions,but were membrane-disruptive at weakly acidic pH.The liposomes were effi-ciently internalized by DCs and delivered OVA into the cytoplasm.In subcutaneous vaccination to mice,the modified liposomes more effectively triggered antigen-specific immune responses than the plain liposomes.OVA vaccination delivered by the modified liposomes significantly inhibited tumor growth and extended the survival of the mice bearing E.G7-OVA tumor.

(4) carboxylated LPG-phosphatidylethanolamine conjugates In theabove studies,pH-sensitive polymers were tightly attached onto the liposome membrane surface by anchoring moieties of long alkyl groups.The alkyl groups might disturb the liposome membrane.Moreover,the tight attachment might discount the pH sensitivity of the liposomes [64].To minimize the unfavorable interaction at neutral pH,new liposome pH-sensitization molecules were further developed by conjugating pH-sensitive polymers 3-methylglutarylated LPG and 2-carboxycyclohexane-1-carboxylated LPG with the phosphatidylethanolamine head group[64],as shown in Fig.4C.In this way,the polymer chains well apart from the liposome surface were expected not to disturb the endosome membrane structures at physiological pH.The liposomes modified with the polymer-lipids were used to deliver antigen for cancer immunotherapy.

6.pH-responsive carriers by using acid-sensitive membrane disruptors

Besides the above carrier design strategies,there are other ways potentially to escape the endosomal/lysosomal systems,for example,by producing gas to explode the membrane systems.Ma et al.[65] designed pH-sensitive poly(D,L-lactic-co-glycolic acid) nanoparticles encapsulating ammonium bicarbonate (NH4HCO3) as antigen OVA carriers by means of both the emulsion-diffusion-extraction and pre-mixed emulsification.The nanoparticles had the size of 893.63 ± 7.93 nm in diameter with low PDI (0.061 ± 0.01),zeta potential(-12.22 ± 2.37 mV),and antigen content of 49.22 ± 2.58 μg OVA/mg particles.The OVA release was enhanced by decomposing NH4HCO3in the weakly acidic condition in DC endosomes/lysosomes.The resulting CO2and NH3broke the nanoparticles,causing rapid antigen release in DC endosomes and lysosomes.Thus,antigens could be released from lysosomes into the cytosol,and be cross-presented to induce cellular immunity.As a result,the immunized mice with the pH responsive nanoparticles induced strong cellular immune response besides antibody protection.The same group [66] further adopted nanoscale CaCO3as an antigen carrier,and prepared hierarchical OVA@CaCO3nanoparticles (～500 nm,-18.8 mV) by using the soft template OVA to guide the CaCO3formation.OVA was entrapped into the particles with a loading ratio of 19.6%.CaCO3nanoparticles could react in the acidic environment of the endosomal/lysosomal systems to produce gas CO2,which could break the systems to release antigens to the cytosol of APCs.The OVA@CaCO3nanoparticles promoted antigen cross-presentation,facilitated the proliferation of CD8+T cells,induced efficient and specific CTL response,and finally achieved promising tumor therapy efficacy.

Besides,membrane-lytic peptides or bacteria pore-forming proteins could also be integrated into cytosol-delivery systems for inducing cross-presentation.For example,Laborde et al.[67] integrated a poreforming protein ─sticholysin (StII) into lysosomes together with antigen OVA to realize antigen cytosol delivery.The liposome/OVA/StII particles exhibited the size of 2.2 ± 0.04 μm,and loaded the two proteins at a similar efficacy for StII(52 ± 8%)and OVA(48 ± 7%),respectively.The mice immunized with the liposome/OVA/StII formulation produced much higher OVA-specific CD8+T cells than those immunized with liposome/OVA formulation.In a prophylactic antitumor test,the mice immunized with the liposome/OVA/StII formulation and then challenged with the E.G7-OVA tumor cells delayed tumor appearance by 10 days,compared with those immunized with liposome/OVA formulation.In addition,Morishita et al.[68] conjugated pH-sensitive fusogenic peptide GALA,(NH2-WEAALAEALAEALAEHLAEALAEALEALAA-COOH),to murine melanoma B16BL6 cells-deriving exsomes.GALA molecules/exosome was measured to be 369,666,and 677 in three different formulations,respectively.The obtained GALA-exsomes with particle size of 106 ± 4 nm and zeta potential of-32.8 ± 1.9 mV,exhibited membrane-lytic activity in acidic environment,and as a result realized efficient cytosolic delivery of exsome tumor antigens.The DCs treated with the GALA-exsomes enhanced the tumor antigen presentation level on MHCⅠmolecules.

7.Conclusion

From the above studies,the cytosolic antigen delivery by the pHresponsive vaccine carriers was mediated mainly by two mechanisms:(1)degradation of the carrier materials in the weakly acidic solution of the endosomes/lysosomes of APCs and then antigen release from the carriers;(2) disruption of the endosome/lysosome membrane by the carrier materials through different mechanisms and then the antigen leakage to the cytosol of APCs.These pH-responsive vaccine carriers displayed outstanding advantages over ordinary vaccine carriers in inducing cellular immunity.It is expectable that more pH-responsive vaccine carriers could be designed based on the above or novel action mechanisms for improving cellular immunity.

8.Perspectives

To promote antigen cross-presentation and cellular immunity,the above design strategies of the pH-responsive antigen carriers bring us many useful inspirations in the development of antigen delivery systems.However,to realize their clinical applications,their respective action mechanisms should be deeply clarified.Some strategies might only stay at the stage of proof of concept and have no clinical application value.Moreover,bio-safety issue of these carriers should be considered.In this sense,non-degradable and/or non-biocompatible carrier materials may have bio-safety risks.Instead,biodegradable and/or biocompatible carrier materials are preferred in terms of their clinical application potential.On the other hand,from biological viewpoint,the occurrence of antigen cross-presentation mainly depends on the cellular internalization pathways of vaccines and their interaction with endosome/lysosome in APCs.Therefore,the basic interdisciplinary research on these two biological processes should be deeply conducted to clarify the mechanisms.This will provide key information to direct the design of antigen carriers that facilitate antigen cross-presentation and finally cellular immune response.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was funded by the National Natural Science Foundation of China (Grant No.31870943).