**3. Evading the immune response**

When gene delivery systems are administered systemically they are usually cleared rapidly from circulation, mainly by Kupffer cells in the liver and macrophages in the spleen. This is a form of defence by the host designed to recognise and clear potentially harmful invaders from the system as quickly as possible, and involves a two-step process initiated by opsonisation with subsequent phagocytosis [71]. Opsonisation is the adsorption of foreign particles by opsonin proteins such as immunoglobulins, blood serum proteins, and complement proteins. Subsequently, macrophages may bind directly to the opsonised particle, engulf and remove it from circulation or the complement system may be activated, also leading to phagocytosis [72]. The characteristics of the particle in circulation play an important role in the recognition process and therefore are extremely important parameters to consider in the design of a delivery vector. Particles larger than the renal threshold of approximately 5,000 Daltons (usually greater than 200 nm hydrodynamic radii) are more likely to activate the complement system and are usually cleared more rapidly than their smaller counterparts. Surface charge, hydrophobicity, and the presence of certain functional groups are also important, with a more cationic nature favouring interaction with the anionic blood proteins and enhancing opsoni‐ sation [73].

Initial opsonisation of particles is critical to their subsequent removal, so if opsonisation can be reduced or avoided, then clearance may be circumvented. An extensively used method to overcome opsonisation is the utilisation of shielding groups or 'stealth' molecules that are generally long hydrophilic polymer chains. These are typically flexible and charge neutral, which can block the electrostatic and hydrophobic interactions between opsonins and the nucleic acid/vehicle complex, improving the stability of the particles in the systemic circula‐ tion. Various polymers have been used including polyacrylamide, poly(vinyl alcohol), poly(Nvinyl-2-pyrrolidone), and poly ethylene glycol (PEG) [72].

PEG is the most commonly used and effective polymer for stealth molecules; PEG is non-toxic, non-immunogenic, non-antigenic, highly water-soluble, and FDA approved. PEGylating a cationic complex shields the positive charge, thereby reducing interaction with blood compo‐ nents, and inhibiting clearance, allowing increased circulation time and opportunity for vectors to reach their target site. It also reduces non-specific binding to non-target cells and stabilises particles, reducing aggregation. The increased circulation time is highly desirable for passive tumour targeting, facilitated by the leaky tumour vasculature. Extravasation of vectors from the blood stream occurs with retention and accumulation in the tumour site by the EPR effect. It has been suggested, however, that repeat administrations may sensitise the immune system to PEG resulting in rapid clearance of PEGylated liposomes from circulation and formation of anti-PEG antibodies [74, 75]. However, the validity of the assays used to test for anti-PEG antibodies have been questioned over flaws and lack of specificity [76].

In order for PEG to properly oppose the attractive forces between the opsonins and the cationic particle surface, it must have a sufficient surface coverage, which is usually correlated to the molecular weight, surface chain density, and conformation of PEG. It is generally held that sufficient stealth character is achieved with a molecular weight of 2,000 Daltons or more, with loss of flexibility in shorter chains being the probable cause for lack of stealth. As molecular weight increases, the blood circulation half-life also increases. Surface chain density and conformation are also important so that adequate surface coverage is achieved to avoid gaps where opsonins may bind, while also maintaining flexibility in the PEG layer responsible for the steric hindrance properties. By fine-tuning such properties of PEG, an improved biodis‐ tribution and the pharmacokinetic profile of the therapeutic may be achieved; such tunings have led to many different PEGylation strategies being developed [72].

#### **3.1. The PEG dilemma**

Anionic plasmid DNA cargo is condensed using a cationic material such as poly-L-lysine or protamine. Vectors are functionalised with adjuncts to aid in evasion of the various barriers that are posed to gene therapy strategies, as highlighted above. The various functional groups

When gene delivery systems are administered systemically they are usually cleared rapidly from circulation, mainly by Kupffer cells in the liver and macrophages in the spleen. This is a form of defence by the host designed to recognise and clear potentially harmful invaders from the system as quickly as possible, and involves a two-step process initiated by opsonisation with subsequent phagocytosis [71]. Opsonisation is the adsorption of foreign particles by opsonin proteins such as immunoglobulins, blood serum proteins, and complement proteins. Subsequently, macrophages may bind directly to the opsonised particle, engulf and remove it from circulation or the complement system may be activated, also leading to phagocytosis [72]. The characteristics of the particle in circulation play an important role in the recognition process and therefore are extremely important parameters to consider in the design of a delivery vector. Particles larger than the renal threshold of approximately 5,000 Daltons (usually greater than 200 nm hydrodynamic radii) are more likely to activate the complement system and are usually cleared more rapidly than their smaller counterparts. Surface charge, hydrophobicity, and the presence of certain functional groups are also important, with a more cationic nature favouring interaction with the anionic blood proteins and enhancing opsoni‐

Initial opsonisation of particles is critical to their subsequent removal, so if opsonisation can be reduced or avoided, then clearance may be circumvented. An extensively used method to overcome opsonisation is the utilisation of shielding groups or 'stealth' molecules that are generally long hydrophilic polymer chains. These are typically flexible and charge neutral, which can block the electrostatic and hydrophobic interactions between opsonins and the nucleic acid/vehicle complex, improving the stability of the particles in the systemic circula‐ tion. Various polymers have been used including polyacrylamide, poly(vinyl alcohol), poly(N-

PEG is the most commonly used and effective polymer for stealth molecules; PEG is non-toxic, non-immunogenic, non-antigenic, highly water-soluble, and FDA approved. PEGylating a cationic complex shields the positive charge, thereby reducing interaction with blood compo‐ nents, and inhibiting clearance, allowing increased circulation time and opportunity for vectors to reach their target site. It also reduces non-specific binding to non-target cells and stabilises particles, reducing aggregation. The increased circulation time is highly desirable for passive tumour targeting, facilitated by the leaky tumour vasculature. Extravasation of vectors from the blood stream occurs with retention and accumulation in the tumour site by the EPR effect. It has been suggested, however, that repeat administrations may sensitise the immune system to PEG resulting in rapid clearance of PEGylated liposomes from circulation and

vinyl-2-pyrrolidone), and poly ethylene glycol (PEG) [72].

will be discussed below.

66 Gene Therapy - Principles and Challenges

sation [73].

**3. Evading the immune response**

A major problem with the use of PEG for cancer gene therapy is that it may hinder gene expression by impeding the entry of the delivery system into tumour cells. The initial inter‐ action of cationic delivery systems with cell membranes relies on electrostatic association, so masking by PEG may have an unfavourable effect. Further to this, the improved stability of PEGylated particles disrupts membrane fusion and may reduce the effects of fusogenic peptides either during cell internalisation or for endosomal disruption. The term 'PEG dilemma' was coined to describe the balance that must be struck between availing of the beneficial characteristics that PEG provides while not being limited by them. Appropriate vector design must ensure that an appropriate balance is struck between the facets that make PEG an attractive supplement to a vector and those that limit its effectiveness [77].

Various strategies have been employed in order to overcome the problems posed by the PEG dilemma. Once the PEGylated vector has survived in circulation and reaches its target cell, the PEG chain becomes redundant. By removal or detachment of PEG from the surface of the vector, interaction with the cell membrane can occur and initiate internalisation. One strategy that has gained much attention is the addition of targeting ligands to PEG that bind to cell surface receptors exclusive to the target cells, limiting endocytosis of the PEGylated delivery system to the target cells [78]. However, this may involve the introduction of a charged moiety onto the PEG, reducing the effectiveness of PEG in evading clearance. The bulky PEG chain may also still hinder the gene delivery system overcoming the various intracellular barriers discussed above. Therefore, the design of a detachable or reversible PEGylation has resulted in 'smart' delivery systems that can exploit different aspects of the intracellular or extracellular tumour environment, including pH, enzyme complement, or reduction, while also functioning as a targeting tool to direct vectors to tumours [79, 80].

#### **3.2. pH-sensitive PEG linkers**

Using linkages such as ester and hydrazine bonds, which are stable in circulation but hydro‐ lysed in acidic conditions, is a promising way of creating a detachable PEG. The acidic tumour microenvironment may cleave off the PEG chain, thereby releasing the therapeutic at the target site and allowing interaction of the cationic delivery system with cell membranes, initiating internalisation. Alternatively, the acidic pH within the endosome may also serve to cleave PEG from the delivery system after receptor-mediated endocytosis. This will unmask the vector allowing endosomal escape by, for example restoring fusogenic activity, facilitating cytosolic delivery and subsequent gene expression [79]. Fella et al. described a targeted polyplex system with PEG attached via an acid labile hydrazone linkage that afforded a 14-fold increase in transgene expression in HUH7 hepatocellular carcinoma tumours compared to the non-acid sensitive formulation. The system was able to protect the vector in the systemic circulation, facilitate entry to the cells via EGF-receptor mediated endocytosis, and exploit endosomal pH to execute the removal of PEG, which permitted release of the vector from the endosome [81].

#### **3.3. Enzymatic cleavage of PEG**

Various proteolytic enzymes are known to be secreted into the extracellular environment by cancer cells. The knowledge of specific enzymes and their substrates can then be exploited to tether PEG to a vector via an appropriate enzyme-cleavable linker. Matrix metalloproteinases (MMPs) are a family of proteases commonly secreted by tumours, degrading the extracellular matrix facilitating growth and progression of tumours [82]. Li et al. took advantage of the presence of MMP-7 proteases in the extracellular environment by functionalising polymeric nanoparticles with PEG via a MMP-7 cleavable linker for delivery of anti-luciferase siRNA. The authors reported a 2.5-fold increase in transfection efficiency in MDA-MB-231 breast cancer cells in the presence of MMP-7 in vitro compared to transfection efficiency in the absence of MMP-7 [83]. These results, however, would need to be further reinforced with in vivo studies in order to fully assess the pharmacokinetic profile of this system.

#### **3.4. Reduction-sensitive PEG linkage**

A reduction-sensitive linkage may be used to attach PEG to a vector using disulphide bonds. These bonds are susceptible to reduction by glutathione (GSH), a peptide with various functions within the cell such as antioxidant defence, metabolic processes, and regulation and maintenance of cellular redox status. The intracellular concentration of GSH is three orders of magnitude higher than in the extracellular compartment [84], which allows for reduction of the disulphide bonds and detachment of the bulky PEG chain once the functionalised vector is inside target cells. Alternatively, extracellular reduction may occur through the action of thiol-containing cell surface receptors [85]. Lei et al. described a targeted delivery system functionalised with PEG attached via a disulphide linkage. Polyethylenimine (PEI) nanopar‐ ticles that were functionalised with a reduction-sensitive linked PEG were twice as potent as their counterparts that lacked the reduction-sensitive linker in terms of GFP and RFP reporter gene delivery in vitro and in vivo in U87 glioblastoma tumours [86].

#### **3.5. Copolymers**

While the use of a cleavable linker for PEGylation has shown promise for gene delivery, issues may arise if the linker is not accessible for cleavage due to the shielding action of PEG. The nature of polymers renders them easily modifiable; changing the characteristics of the PEG polymer itself by forming crosslinks may produce a degradable copolymer suitable for controlled release [79]. Fan et al. reported on a copolymer that comprised polyethylene glycol 5000 (PEG114), Vitamin E (VE), and thioctic acid (TA), termed PEG114:VE:TA, which assem‐ bled into micelles with poly-disulfide crosslinks [87]. The copolymer resulted in improved thermodynamic and kinetic profile of the anticancer drug paclitaxel. Reduction of the disul‐ phide crosslinks occurred in response to glutathione causing rapid disassembly of the micelles and accelerated drug release that resulted in approximately 3-fold higher plasma concentration than the non-crosslinked micelles leading to increased drug accumulation in the SKOV-3 human ovarian cancer xenograft mouse model. Although this study did not deliver gene therapy, it demonstrates the potential of modifying polymer crosslinks to achieve desirable characteristics for drug delivery.

site and allowing interaction of the cationic delivery system with cell membranes, initiating internalisation. Alternatively, the acidic pH within the endosome may also serve to cleave PEG from the delivery system after receptor-mediated endocytosis. This will unmask the vector allowing endosomal escape by, for example restoring fusogenic activity, facilitating cytosolic delivery and subsequent gene expression [79]. Fella et al. described a targeted polyplex system with PEG attached via an acid labile hydrazone linkage that afforded a 14-fold increase in transgene expression in HUH7 hepatocellular carcinoma tumours compared to the non-acid sensitive formulation. The system was able to protect the vector in the systemic circulation, facilitate entry to the cells via EGF-receptor mediated endocytosis, and exploit endosomal pH to execute the removal of PEG, which permitted release of the vector from the endosome [81].

Various proteolytic enzymes are known to be secreted into the extracellular environment by cancer cells. The knowledge of specific enzymes and their substrates can then be exploited to tether PEG to a vector via an appropriate enzyme-cleavable linker. Matrix metalloproteinases (MMPs) are a family of proteases commonly secreted by tumours, degrading the extracellular matrix facilitating growth and progression of tumours [82]. Li et al. took advantage of the presence of MMP-7 proteases in the extracellular environment by functionalising polymeric nanoparticles with PEG via a MMP-7 cleavable linker for delivery of anti-luciferase siRNA. The authors reported a 2.5-fold increase in transfection efficiency in MDA-MB-231 breast cancer cells in the presence of MMP-7 in vitro compared to transfection efficiency in the absence of MMP-7 [83]. These results, however, would need to be further reinforced with in vivo studies

A reduction-sensitive linkage may be used to attach PEG to a vector using disulphide bonds. These bonds are susceptible to reduction by glutathione (GSH), a peptide with various functions within the cell such as antioxidant defence, metabolic processes, and regulation and maintenance of cellular redox status. The intracellular concentration of GSH is three orders of magnitude higher than in the extracellular compartment [84], which allows for reduction of the disulphide bonds and detachment of the bulky PEG chain once the functionalised vector is inside target cells. Alternatively, extracellular reduction may occur through the action of thiol-containing cell surface receptors [85]. Lei et al. described a targeted delivery system functionalised with PEG attached via a disulphide linkage. Polyethylenimine (PEI) nanopar‐ ticles that were functionalised with a reduction-sensitive linked PEG were twice as potent as their counterparts that lacked the reduction-sensitive linker in terms of GFP and RFP reporter

While the use of a cleavable linker for PEGylation has shown promise for gene delivery, issues may arise if the linker is not accessible for cleavage due to the shielding action of PEG. The nature of polymers renders them easily modifiable; changing the characteristics of the PEG

in order to fully assess the pharmacokinetic profile of this system.

gene delivery in vitro and in vivo in U87 glioblastoma tumours [86].

**3.3. Enzymatic cleavage of PEG**

68 Gene Therapy - Principles and Challenges

**3.4. Reduction-sensitive PEG linkage**

**3.5. Copolymers**

The production of copolymers, which combine the characteristics of more than one polymer, has shown promise where a balance is struck between PEGylation and copolymer reducible characteristics. Recently, Lai et al. presented a reducible copolymer comprising poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-SS-P[Asp(DET)] (P(EPE)-SS-P[Asp(DET)]), which possesses a redox potential-sensitive disulfide linkage between the P(EPE) polymer and the cationic block P[Asp(DET)]. The copolymer was used to deliver the pGL4 DNA vector for luciferase expression, and a 2-fold increase in transfection efficiency was observed compared to delivery with non-reducible copolymer counterparts in MDA-MB-231 breast cancer cells in vitro [88]. However, much work is yet to be done to elucidate the exact characteristics and polymer design needed to produce optimal transfection efficiencies.

Systems that combine two or more mechanisms for masking delivery vectors while improving uptake have been investigated, with PEG being combined with other polymers or peptides. Huang et al. designed a multifunctional delivery system that uses a combination of an MMPsensitive linkage, a pH-sensitive mask to quench the cationic charge of nona-arginine (R9), and PEG to improve steric stabilisation in circulation [89]. The masking peptide was pH-sensitive with an isoelectric point (pI) of 6.4, affording the masking peptide a negative charge at physiological pH, which interacts with the cationic R9 cell-penetrating peptide (CPP). How‐ ever, a tumour's acidic environment neutralises the masking peptide, allowing the cationic nature of R9 to come to the fore. Cleavage of the PEG by MMP-2 allowed the CPP-cargo complex to enter cells. The authors used in vivo imaging to demonstrate the specificity of these nanoparticles to target human hepatocellular carcinoma cell (BEL-7402) xenografts.

Although PEGylation provides a means of enhancing circulation times, allowing vectors to reach their target site, reliance on the EPR effect as a means of passive targeting may not be as reliable as initially thought. The variability displayed in tumour biology as well as the disordered and discontinuous tumour vascular structure means that the accumulation of delivery vectors by the EPR effect may not give a tumour-wide distribution. Although it may give an added advantage, total reliance on the EPR effect cannot give reliable results and so there is a need for an active targeting strategy [90].
