The coating polymer can be attached to a lipid anchor, which allows grafting to a lipidic surface, e.g. liposomal bilayers. A linker with a predetermined cleavage point between the polymer chains and the anchoring moiety can be introduced in this polymer–lipid conjugate. Chemical stimuli, such as the presence of low pH or reducing agents, and enzymatic stimuli, such as proteases, can induce cleavage of the linker. For an overview of shedding approaches using cleavable linkers see Table .
Low pH-Induced Shedding
After entry into the target cell via receptor-mediated endocytosis, which is the uptake process for most particulate carriers, a lower pH is encountered in the endo/lysosomal compartment (24
). Endosomes display a pH around 5.5-6.0 and lysosomes around 4.5. The extracellular pH within pathological sites such as tumors, inflammatory tissue and myocardial ischemia can also be lower than the physiological pH (25
). If pH-sensitive functional groups are applied as linkers between the polymer coating and its anchor, they may undergo protonation in the low pH environment, leading to hydrolysis of the sensitive bond and therefore to detachment of the polymer molecule. For nanoparticulate systems the diorthoester, the orthoester, the vinyl ether, the phosphoramidate, the hydrazone, and the β-thiopropionate linker have been applied as acid-sensitive linkers. An overview of the hydrolysis reactions involved is given in Fig. . Besides covalent bonds, linking of the coating to the carrier by electrostatic attraction, which are reduced or even abrogated at lower pH, has been pursued.
Hydrolysis reactions relevant to acid-sensitive linkers used in sheddable coatings. R1 contains the hydrophilic part of the molecule (PEG), R2 contains the hydrophobic anchor, ODN or PLL, depending on the type of carrier.
The diorthoester linker
The diorthoester bond (Fig. a) is one of the most pH-sensitive functional groups. Orthoesters are relatively stable under neutral conditions, but upon protonation they hydrolyze quickly due to the formation of a stabilized dialkoxy carbonium intermediate (28
). The group of Szoka focused on the design of a sheddable coating which stabilizes the fusogenic DOPE in liposomes at neutral pH. PEG was linked to distearoyl glycerol via a diorthoester bond (PEG2000
-diortho ester–distearoyl glycerol (POD) conjugate). Hydrolysis of this bond at the low endosomal pH was expected to result in destabilization of the DOPE-liposomes by promoting
phase transition and thereby to mediate fusion with the endosomal membrane and contents release into the cytosol (29
). While most of the POD conjugate remained intact over 3 h of incubation at 37°C and pH 7.4, complete degradation occurred within 1 h at pH 4 and 5. A fluorescence quenching assay was used to study the leakage and thereby destabilization of POD-liposomes. Liposomes with 10 mol% of POD were stable in alkaline buffer for 2 weeks (pH 8.5). At pH 5-6, resembling endosomal conditions, the liposomes collapsed within 10–100 min, showing extensive aggregation and contents leakage. Increasing amounts of POD on the liposome surface resulted in slower leakage kinetics (30
). Kinetic studies in mice were performed with the POD/DOPE liposomes and revealed a half-life of 194 min (29
). In comparison, PEG/DOPE liposomes had a half-life of 295 min. Based on the results of the leakage and lipid mixing assays, a ‘minimum surface shielding model’ was proposed (31
). Two phases of release were observed: an initial lag phase with only little leakage and a burst phase with extensive leakage. Upon pH decrease, hydrolysis of the diorthoester bond results in a reduction of the amount of POD molecules on the liposome surface (lag phase). Below a certain threshold level of surface-attached POD, intervesicular mixing occurs and contents release is drastically intensified (burst phase). The advantage of the use of the POD conjugate is that rapid hydrolysis occurs at a mildly acidic pH. This property makes the POD conjugate especially attractive for drug delivery situations in sites where the pH reduction is only one pH unit or less, such as in inflamed or malignant tissue.Besides application for liposomes, the use of POD as a sterically-stabilizing coating has been studied for nonviral gene delivery vectors, in this particular case lipoplexes (32
). Cellular uptake of these particles is mediated by electrostatic interaction of the positively charged lipoplexes with the negatively charged cell membranes. A cleavable PEG-conjugate attached to the surface of these carriers should shield the positive charge in the circulation and thereby reduce unspecific interactions with blood components and increase the circulation half-life. On the other hand, the cleavable PEG coating should still allow successful delivery of the DNA and subsequent protein expression. To achieve this, stabilized plasmid-lipid particles (SPLP), composed of the cationic lipid 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), DOPE, POD and plasmid DNA were designed. Shedding of the PEG-coating was assessed by studying the pH-and time-dependent collapse of the particles using dynamic light scattering. While pH-insensitive PEG-SPLP did not collapse at low pH, POD-SPLP with 13 mol% POD collapsed within 110 min at pH 5.3, as evidenced by an increase of the mean diameter of several hundreds of nanometers. PEG-SPLP and POD-SPLP were taken up by cultured CV-1 cells to a similar extent and in a similar punctuate pattern, but only the latter showed considerable gene transfection. This was likely a result of a faster endosomal escape mediated by shedding of the coating. POD nanoparticles with encapsulated plasmid DNA (referred to as NLP) showed superior transfection properties in CV-1 cells as compared to a pH-insensitive control formulation (50–100 times higher transfection levels) (30
). This was attributed to an enhanced endosomal escape of the DNA of these formulations, mediated by shedding of the coating. The authors claimed the POD formulation (POD with PEG2000
) as the most optimal formulation when compared to formulations with diorthoester conjugates with a PEG molecular weight of 750 and 5,000, regarding small size, stability, cytotoxicity, and transfection efficiency.
The orthoester linker
Besides the diorthoester bond, also the orthoester bond (Fig. b) has been studied for its applicability to mediate shedding. Four PEG–orthoester lipid conjugates composed of either an octadecyl alkyl chain or a cholesteryl group (hydrophobic anchor) and a PEG5000
chain linked by either a six-membered or a five-membered orthoester ring structure were synthesized by Masson et al.
). To assess the shedding potential, hydrolysis rates of these conjugates were determined. At room temperature and pH 7.5, conjugates were stable for several days. Hydrolysis at pH 5 was the fastest for the conjugate with the cholesteryl group and the six-membered ring orthoester and complete within 1 h. Conjugates were used for the steric stabilization of lipoplexes based on a cationic lipopolyamine, DOPE, and plasmid DNA. Particle destabilization rates at lower pH were highest when the six-membered ring orthoester conjugate with the cholesteryl group was used. At pH 5 lipoplexes prepared with this conjugate aggregated within 30 min. Transfection studies in vitro
revealed a 25-fold increase in transfection of HeLa cells in case of lipoplexes prepared with the most optimal PEG-orthoester lipid conjugate as compared to lipoplexes coated with non-cleavable PEG.
The vinyl ether linker
The vinyl ether linkage (Fig. c) was exploited by the group of Thompson to synthesize different vinyl ether-linked PEG–lipid conjugates and develop a shedding strategy for DOPE-based liposomes (34
). Upon protonation of the vinyl ether β-carbon of this functional group, the double bond is cleaved releasing an alcohol and an aldehyde. PEG of two chain lengths (PEG2000
) was coupled to 1,2-dioleyl-rac
-glycerol (DOG) or DOPE as the lipid anchor, respectively. To evaluate shedding, the potential of these conjugates to promote low pH-induced calcein release was investigated. Calcein was released from the liposomes within the time scale of hours in an acidic environment (pH
5). For example, the formulation with 5 mol% of the PEG2000
-vinyl ether–DOPE conjugate showed only 14% calcein release at pH 7.4, whereas 95% of the calcein was released at pH 3.5. This nearly complete release, however, required 48 h of incubation. The rate of shedding increased in the case of the DOPE-anchor systems as the surface was negatively charged and therefore a higher proton concentration is present at the carrier surface. Furthermore, the vinyl ether bond orientation with respect to the membrane bilayer likely played a critical role, as shedding occurs more slowly if the sensitive linker is oriented closer to the hydrophobic acyl chain region.The vinyl ether bond was also used to link PEG5000
to a hydrogenated cholesterol moiety (DHCh-MPEG5000
). Hydrolysis of the conjugate was slow with the reaction still incomplete after 5 days at pH 4.5. Studies on the interaction of fusogenic DOPE/DHCh-MPEG5000
liposomes with endosome-like membranes suggested that even upon prolonged exposure to a low pH, fusion with endosome-like membranes did not occur. The authors conclude from their findings that the slow cleavage rate of the conjugate is insufficient to make it applicable for shedding in vivo
.Improved properties of vinyl ether-linked PEG derivatives were demonstrated in a subsequent publication (36
). A PEG5000
-derivatized analogue of diplasmenylcholine with a vinyl ether linkage ((R
-methoxy-poly(ethylene glycolate)), BVEP) was synthesized and utilized to stabilize DOPE liposomes (37
). The shedding ability was assessed by analysis of the hydrolysis kinetics of BVEP, dye release from BVEP/DOPE liposomes and liposome fusion at low pH values. Eighty percent of the BVEP was hydrolyzed at pH 4 after 60 min. Liposomes containing 1-5 mol% of the BVEP showed only little calcein release at neutral pH in buffer for up to one day. Also in 10% serum, these formulations were relatively stable (approx. 20% calcein leakage over 20 h) (38
). The most optimal liposome formulation regarding dye retention at neutral pH and dye release at acidic pH (BVEP/DOPE 3:97) showed a collapse at pH 4.5 within 3 h as shown by c-TEM. However, at this pH calcein release kinetics (78% after 24 h) and levels of membrane fusion (5% after 72 h) were low (36
). Several explanations for the slow kinetics were proposed: a generally slow hydrolysis rate, alterations of the microenvironment at the hydrolysis site (e.g. a less acidic pH than in the surrounding solution), and an inhibition of the DOPE phase transition caused by e.g. retention of hydrolyzed PEG on the liposome surface. The authors conclude that contents release and membrane fusion were not rapid enough and must be increased for application in vivo
The phosphoramidate linker
The acid-induced shedding approach was also applied to DNA/PEG hybrid micelles for delivering ODN, which are able to bind to cytoplasmic mRNA to block specific gene expression (39
). Shedding of the stabilizing PEG layer can be advantageous as PEG might sterically hinder interaction of ODN with the target RNA after ODN-PEG have been released into the cytosol. Jeong et al.
used an acid-cleavable phosphoramidate group (Fig. d) to covalently link the ODN to PEG resulting in a diblock copolymer-like structure. At a pH of 4.7, almost quantitative linkage hydrolysis was achieved within 5 h. The negatively charged ODN–PEG conjugate was complexed with the cationic fusogenic peptide KALA to increase endosomal escape. This resulted in polyelectrolyte complex micelles with the PEG molecules forming the hydrophilic corona. A comparison with a formulation bearing PEG with a non-cleavable linker was not performed. Thus, although the results on the delivery and antisense effect found in this study were encouraging, they provide no evidence for a benefit of the PEG shedding.
The hydrazone linker
Another linker that is sensitive to low pH and therefore of potential value for use in sterically stabilized nanoparticle systems is the hydrazone bond (Fig. e). Sawant et al.
designed micelles and liposomes with a sheddable coating using the pH-sensitive hydrazone linker to couple PEG to phosphatidylethanolamine (PEG-Hz-PE) (40
). The stability of PEG-Hz-PE micelles was assessed by size exclusion chromatography. The micelles were intact after 20 min of incubation at room temperature at pH 10, whereas at pH 7 the amount of micelles was reduced to 56% and at pH 5 only 3% of the micelles were recovered. PEG-Hz-PE micelles composed of PEG-PE (40 mol%) and PEG-Hz-PE (54 mol%) were prepared. Biotin was included as a model targeting ligand to be exposed after shedding. Biotin binding was assessed on a NeutrAvidin affinity column. While at pH 8 only 15% of the micelles were retained on the column, 75% of the micelles were retained after 15 min incubation at pH 5, due to exposure of the biotin ligand after shedding of the PEG-coating. PEG-Hz-PE carriers were tested for association and internalization by 3T3 cells (micelles) and U-87 MG cells (liposomes) using fluorescence microscopy. For these experiments, cell penetrating TAT moieties were used instead of biotin groups. Micelles with 5 mol% of the TAT peptide were only marginally associated with cells when kept at pH 8, however association was clearly enhanced when micelles were preincubated for 20–30 min at pH 5 before adding them to cells. For liposomes with 9 mol% of the cleavable PEG-Hz-PE, association with cells were at levels comparable to the non-coated liposomes after preincubation for 20 min at pH 5, indicating that the TAT function could mediate association after shedding.Walker and colleagues have reported on the synthesis of several different poly-l
-lysine (PLL)–PEG conjugates linked via hydrazone bonds for non-viral gene delivery systems (41
). The two most promising candidates showed hydrolysis half-lives of 30 min (acylhydrazone-linked PEG-conjugate) and 1.5 h (2-pyridylhydrazone-linked PEG-conjugate) at pH 7.4 and 37°C, but only 10% of the conjugates remained intact after 10 min at pH 5 and 37°C. Shedding was assessed by measuring changes in zeta potential and particle size of the polyplexes. Polyplexes prepared without PEG-conjugates aggregated after 1 h at 37°C in the presence of salt. Polyplexes with the 2-pyridylhydrazone-linked and the acylhydrazone-linked conjugates were stable for 5 h and 30 min of incubation at pH 7.4 and 37°C, respectively. However, at pH 5, within two hours (2-pyridylhydrazone-linked conjugate) and 30 min (acylhydrazone-linked conjugate), a maximum in surface charge and aggregation of polyplexes was reached. Transfection studies with poly (ethylene imine) (PEI)-based polyplexes shielded with hydrolysable PEG-conjugates showed that transfection levels in different cell types were similar to those obtained with non-shielded polyplexes and were approximately 40–100 fold higher than for a polyplex formulation with a non-hydrolysable PEG-PLL conjugate. The authors assume that an improved endosomal escape of the DNA after shedding of the PEG-coating is responsible for improved transfection activity. In a subcutaneous mouse tumor model, epidermal growth factor receptor (EGF) targeted polyplexes were used for transfection after intravenous administration. Polyplexes prepared with the 2-pyridylhydrazone-linked conjugate showed more than one order of magnitude higher luciferase expression in the tumor when compared to polyplexes with a non-hydrolysable PEG-PLL conjugate. The authors suggest that shedding increases the intracellular trafficking efficiency of the polyplexes by increasing for example endosomal escape.
The β-thiopropionate linker
The group of Kataoka investigated a β-thiopropionate linker (Fig. f) as acid-labile ester group to link antisense ODN to PEG and form polyion complex (PIC) micelles with these conjugates together with linear PEI (42
). At pH 7.4 almost no cleavage of the linker was observed, however, 97% was cleaved after 24 h at pH 5.5. Size exclusion chromatography showed that at pH 5.5 the PIC micelle peak completely disappeared after six hours. Precipitation was observed in the samples. Micelles with the β-thiopropionate linker had an increased antisense effect as compared to micelles with an acid-insensitive linker (65% vs. 27% inhibition of luciferase gene expression) (43
). The authors conclude that the cleavage of the acid-labile linker in the endosomes is an important requirement for the release of the free ODN into the cytosol and subsequent antisense effect. Besides ODN-mediated effects, successful gene silencing was achieved with PIC micelles prepared by coupling siRNA to PEG via the β-thiopropionate linker and complexation with PLL (44
Linking via electrostatic interactions
In addition to covalent bonds, also non-covalent bonds have been exploited for low-pH induced shedding. Finsinger and colleagues designed PEG-peptide conjugates, containing negatively charged glutamic acid residues, which bind electrostatically to positively charged PEI-DNA complexes (45
). These conjugates reduced opsonization as compared to non-coated polyplexes and were designed to dissociate from the PEI-DNA complex at the low pH in endosomes due to protonation of the negatively charged peptides. They were equally effective in in vitro
gene delivery when compared to non-coated polyplexes. A similar approach was pursued by Auguste et al.
). Different PEG-polycation copolymers were synthesized and adsorbed onto liposomes containing the pH-sensitive lipid 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), which is neutral at pH 7.4 and positively charged at pH 5.5. Lowering the pH from 7.4 to 5.5 was expected to result in a positively charged liposome surface and consequently desorption of the PEG-polycation copolymers from the liposome surface. Indeed, copolymers with PEG5000
-pDMAEMA showed decreased association constants upon lowering the pH from 7.4 to 5.5.Sethuraman et al.
designed poly(methacryloyl sulfadimethoxine)-block-PEG (PSD-b-PEG) copolymers, which were adsorbed onto a positively charged DNA/PEI complex at neutral pH due to the negatively charged PSD (47
). Size and zeta potential analysis of DNA/PEI/PEG-b-PSD polyplexes showed changes at a pH around 6.6, indicating neutralization of PSD and detaching of the PEG-b-PSD copolymer from the complex surface. Cell viability experiments showed that polyplexes were non-toxic at pH 7.4, but that cell viability was decreased to 20% at pH 6.6. Transfection studies showed a 10-fold increase in transfection when altering the pH from 7.4 to 6.6. The authors noted that a disadvantage of this system is that the detached copolymers are nondegradable and the aggregates formed may be toxic.Acid-triggered shedding represents a straightforward approach. Among the acid-releasable coatings investigated so far, the diorthoester and the hydrazone-linked coatings are probably the most advanced. The diorthoester linkage has fast hydrolysis kinetics and a relatively mild pH decrease already induces shedding (29
). That makes the system most attractive for extracellular shedding in tumors and inflammatory tissue.
Bioconjugation using reduction-sensitive linkers has been a popular approach in the design of drug delivery systems (for a review see (48
)). The disulfide bond (-S-S-) is reversible, but relatively stable in plasma. This bond is formed by oxidation of two sulfhydryl groups and can be cleaved by reducing agents. Extracellularly, due to the oxidizing environment, formation or maintenance of the disulfide bond is favored. The high cytosolic concentration of sulfhydryl group-carrying molecules (e.g. glutathion) creates a reductive environment and cleavage of the disulfide bond occurs relatively quickly under these conditions. Moreover, reduction of these bonds can take place at the cell surface catalyzed by cell surface-associated redox enzymes and in the endo/lysosomal compartment due to the presence of e.g. cysteine and redox enzymes. Therefore, for particulate carriers bearing a sheddable coating that responds to changes in the redox potential, improved cellular interaction, enhanced endosomal escape, and/or cytosolic destabilization of the carrier can be envisaged. Molecular structures of the conjugates used as coatings to achieve reduction-induced shedding are summarized in Fig. .
Reduction-sensitive PEG–S–S–lipid conjugates.
Zalipsky and colleagues stabilized DOPE liposomes by incorporation of 3-6 mol% of a disulfide-linked PEG conjugate. PEG was coupled to distearoyl phosphatidylethanolamine (DSPE) via 3,3′-dithiopropionate (DTP) as linking moiety (PEG-DTP-DSPE, Fig. a) (49
). Shedding was evaluated by dye release and lipid mixing studies. No leakage of liposome contents occurred upon incubation in buffer (pH 7.2) or serum at 37°C for 36 h. Yet, in the presence of 10 mM dithiothreitol (DTT), a complete release of the encapsulated dye and lipid mixing was observed (50% release and lipid mixing within approximately one hour), likely as a result of shedding and subsequent liposome destabilization. DOPE/CHEMS/PEG-DTP-DSPE liposomes (cholesteryl hemisuccinate (CHEMS) was included to achieve pH sensitivity), retained an encapsulated dye in buffer of pH 5.5 at 37°C during at least one day. When DTT or cell-free extracts to mimic intracellular environments were added, liposomes released their contents within a time-scale of hours (49
). After detachment of the PEG chains, the phospholipid derivative 3-mercaptopropionyl-DSPE, was retained on the liposome surface, which might hinder contact between bilayers of the fusogenic liposomes and therefore be responsible for additional stabilization (49
). In vitro
toxicity and nuclear accumulation studies of doxorubicin (DXR) loaded liposomes showed that formulations with the cleavable PEG conjugate were not more cytotoxic than the formulation with the non-cleavable PEG conjugate. Nevertheless, a faster nuclear accumulation of DXR over a period of 12 h was seen for the PEG-DTP-DSPE containing formulation (50
). Survival studies performed in a murine model of B-cell lymphoma demonstrated an improved therapeutic efficacy of doxorubicin encapsulated in anti CD19-targeted DOPE/CHEMS/PEG-DTP-DSPE liposomes as compared to the non-cleavable formulation at the same doxorubicin dose (1.5 times increased life span in mice). Surprisingly, DOPE-based liposomes stabilized with up to 8 mol% of the cleavable PEG conjugate did not yield improvement of circulation times in mice when compared to non-coated liposomes, possibly caused by rapid cleavage of the disulfide bridges in the circulation.
To avoid this rapid cleavage, a dithio-3-hexanol linker was evaluated to link PEG to 1,2-distearoyl-sn
-glycerol-3-phosphatidic acid (PEG-DTH-DSPA, Fig. b) (51
). The attack of the DTH linker is sterically hindered by a propyl group, which was expected to result in a higher stability of the disulfide bond. And indeed, PEG-DTH-DSPA containing liposomes showed similarly prolonged circulation times as compared to liposomes coated with non-cleavable PEG. Studies on release of doxorubicin in the presence of 5 mM DTT showed that all PEG-coated formulations released the drug relatively slowly, in a time scale of hours. An exception was the formulation with 15 mol% of PEG-DTH-DSPA, which showed enhanced leakage as compared to the non-cleavable formulation at pH 7.4, likely mediated by shedding of the PEG-coating: approximately 90% of the liposomal DXR was released over 24 h of incubation in case of the cleavable formulation versus less than 25% release in the case of the non-cleavable one.
To improve the reductive shedding approach, an alternative class of reduction-sensitive linkers to couple PEG to DSPE was designed (Fig. c) (52
). Dithiobenzyl carbamate linkers (DTB, in o
- and p
- configurations) were expected to be cleaved under mild thiolytic conditions due to their mixed aliphatic and aromatic nature. Furthermore, as an amine endgroup was generated after cleavage rather than the bulky mercaptopropionyl derivative in case of the DTP linker, an improved restoration of fusogenicity after shedding of the coating was expected. The conjugates were susceptible to cleavage in the time scale of minutes in an already mild reductive environment (150 μM cysteine) as shown by HPLC analysis, even when attached to a liposome surface. When 3 mol% of PEG-DTB-DSPE was incorporated into DOPE liposomes, this formulation showed no contents release in buffer at pH 7.2 and 37°C within at least 60 min of incubation, but did release an encapsulated dye within 60 min when mild reducing conditions were applied. After prolonged exposure to a low concentration of 15 μM cysteine, which is the average concentration found in plasma, a slight content release was also observed (10-20% within 60 min), indicating that the conjugates were possibly even too trigger-sensitive and that drug release may already take place during circulation.
A different shedding approach was pursued by Maeda et al.
). To avoid drug inactivation in the endo/lysosomal compartment, fusogenic liposomes with a reduction-sensitive sheddable PEG-coating and R8 cell-penetrating peptide coupled to the liposome surface were designed. Shedding of part of the PEG molecules was proposed to occur in the vicinity of the cell surface and should lead to exposure of the cell-penetrating R8 peptide, which would promote uptake by plasma membrane translocation. Further removal of the PEG-coating in the cytosol was proposed to lead to destabilization of liposomes and consequently drug release. A PEG-S-S-DOPE conjugate (Fig. d) was synthesized and incorporated into calcein containing phosphatidylcholine (PC)/DOPE/CHEMS/R8 liposomes at a level of 5 mol%. In the absence of a reducing agent, calcein was not released within 24 h. In the presence of 30 mM cysteine, only approximately 10% release was observed after 24 h. With 240 mM cysteine a maximum release of 30% was reached after 7 h, with 10 mM glutathione release was fast, but incomplete (approximately 70% release within 4 h). Internalization of calcein-loaded liposomes was studied using A431 cells. Liposomes with the cleavable PEG-coating were internalized in a cysteine concentration-dependent manner. Relatively low cysteine concentrations of ≤ 30 mM, for which no calcein release was observed, resulted in internalization of the carrier. Furthermore, it was demonstrated that cysteine was essential for plasmid delivery and transfection. The authors conclude that at mild reducing conditions, which may be encountered at the potential target sites, part of the PEG molecules are cleaved off with display of the R8 peptide as a result. After arrival in the cytosol, destabilization of the carrier can occur at the higher concentrations of reducing agents encountered.
Results of Carlisle et al.
show the first successful reduction-induced shedding approach for polyplexes (54
). DNA/PEI complexes with thiol groups were surface-coated with pyridyldisulfanyl groups-containing polyHPMA as a ‘stealth’ coating, generating a disulfide linker between PEI and polyHPMA. Polyplexes did not release DNA upon incubation with a polyanion, indicating sufficient stabilization. In the presence of 20 or 40 mM DTT, however, DNA was released after 2 hrs of incubation. Transfection activities of polyplexes with the reducible linker were 40-fold higher than those obtained with polyplexes with a non-cleavable linker. Increasing cytosolic reducing activity by co-delivery of glutathion monoethyl ester resulted in increased transfection levels, whereas co-delivery of a glutathion-inhibitor decreased transfection levels, demonstrating a clear role for shedding in this approach. The applicability of using polyHPMA to prolong polyplex circulation times in vivo
, however, remains to be evaluated.
Shedding triggered by high redox-potentials in tissues is an appealing strategy, as demonstrated by the various research groups (49
). However, often high concentrations of reductive agents are required which are not present in physiological/pathological environments (49
). Nanoparticles are mainly taken up by endocytosis, however, disulfide reduction is a slow process under the acidic conditions encountered in the endo/lysosomal compartment (55
). The cytosol is the location with the highest concentration of reducing agents the carrier can encounter and therefore the best shedding site. Particles which are taken up by endocytosis, however, might not reach the cytosol and therefore the applicability of these systems might be limited.