Factors influencing retention of adenovirus within tumours following direct intratumoural injection.

Direct intratumoural (IT) administration of adenovirus is widely used, however little is known about the resulting distribution of virus particles. Here we have evaluated the influence of tumour size, volume of injectate and occlusion of injection sites (to prevent retrograde seepage) on particle biodistribution and transgene expression. In subcutaneous MDA-231 xenografts, IT injection of relatively large volumes (4 x 20% (vol/vol) injections) resulted in just 40% of the administered dose being retained in tumour tissue after 30 min, with 15% in the liver thought to reflect systemic 'overflow'. Occlusion of the injection sites using surgical adhesive increased retention of the vector to 80% in the tumour with no increase in liver levels. Spread of expression was enhanced using multiple injection sites, but not by using larger injectate volumes. In ZR75.1 breast carcinoma xenografts virus distribution was different, with no evidence of systemic overflow leading to hepatic transduction following IT injection. Typically, clinical doses employ up to 30% vol/vol IT injections. Depending on the tumour, this may give considerable systemic overflow and might account for the high frequency of fevers observed. Virus performance might be improved by tailoring volumes and frequency of IT injection for tumour biology or histotype.

Gene therapy targeting to tumor endothelium.

Tumor-associated vasculature is a relatively accessible component of solid cancers that is essential for tumor survival and growth, providing a vulnerable target for cancer gene therapy administered by intravenous injection. Several features of tumor-associated vasculature are different from normal vasculature, including overexpression of receptors for angiogenic growth factors, markers of vasculogenesis, upregulation of coagulation cascades, aberrant expression of adhesion molecules and molecular consequences of hypoxia. Many of these differences provide candidate targets for tumor-selective 'transductional targeting' of genetically- or chemically modified vectors and upregulated gene expression can also enable 'transcriptional targeting', regulating tumor endothelia-selective expression of transgenes following nonspecific gene delivery. Tumor vasculature also represents an important site of therapeutic action by the secreted products of antiangiogenic gene therapies that are expressed in non-endothelial cells. In this review we assess the challenges faced and the vectors that may be suitable for gene delivery to exploit these targets. We also overview some of the strategies that have been developed to date and highlight the most promising areas of research.

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via alpha6-integrins.

Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.

Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells.

Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.

Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA.

To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5-15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5-50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2+/-5.9-45.1+/-3.9 mV) than those formed using 100 mol% TMAEM (57.8+/-8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3%; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery.

Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers.

Complexes formed between DNA and cationic polymers are attracting increasing attention as novel synthetic vectors for delivery of genes. We are trying to improve biological properties of such complexes by oriented self-assembly of DNA with cationic-hydrophilic block copolymers, designed to enshroud the complex within a protective hydrophilic polymer corona. Poly(L-lysine) (pLL) grafted with range of hydrophilic polymer blocks, including poly(ethylene glycol) (pEG), dextran and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA), shows efficient binding to DNA and mediates particle self-assembly and inhibition of ethidium bromide/DNA fluorescence. The complexes formed are discrete and typically about 100 nm diameter, viewed by atomic force microscopy. Surface charges are slightly shielded by the presence of the hydrophilic polymer, and complexes generally show decreased cytotoxicity compared with simple pLL/DNA complexes. pEG-containing complexes show increased transfection activity against cells in vitro. Complexes formed with all polymer conjugates showed greater aqueous solubility than simple pLL/DNA complexes, particularly at charge neutrality. These materials appear to have the ability to regulate the physicochemical and biological properties of polycation/DNA complexes, and should find important applications in packaging of nucleic acids for specific biological applications.

Nanotherapeutics shielded with a pH responsive polymeric layer.

Efficient intravenous delivery is the greatest single hurdle, with most nanotherapeutics frequently found to be unstable in the harsh conditions of the bloodstream. In the case of nanotherapeutics for gene delivery, viral vectors are often avidly recognized by both the innate and the adaptive immune systems. So, most modern delivery systems have benefited from being coated with hydrophilic polymers. Self-assembling delivery systems can achieve both steric and lateral stabilization following surface coating, endowing them with much improved systemic circulation properties and better access to disseminated targets; similarly, gene delivery viral vectors can be 'stealthed' and their physical properties modulated by surface coating. Polymers that start degrading under acidic conditions are increasingly investigated as a pathway to trigger the release of drugs or genes once the carrier reaches a slightly acidic tumor environment or after the carrier has been taken up by cells, resulting in the localization of the polymer in acidic endosomes and lysosomes. Advances in the design of acid-degradable drug and gene delivery systems have been focused and discussed in this article with stress placed on HPMA-based copolymers. We designed a system that is able to "throw away" the polymer coat after successful transport of the vector into a target cell. Initial biological studies were performed and it was demonstrated that this principle is applicable for real adenoviral vectors. It was shown that the transfection ability of coated virus at pH 7.4 is 75 times lower then transfection at pH 5.4.

Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block co-polymers.

Cationic polymers can self-assemble with DNA to form polyelectrolyte complexes capable of gene delivery, although biocompatibility of the complexes is generally limited. Here we have used A-B type cationic-hydrophilic block co-polymers to introduce a protective surface hydrophilic shielding following oriented self-assembly with DNA. Block co-polymers of poly(ethylene glycol)-poly-L-lysine (pEG-pLL) and poly-N-(2-hydroxypropyl)methacrylamide-poly(trimethylammonioethyl methacrylate chloride) (pHPMA-pTMAEM) both show spontaneous formation of complexes with DNA. Surface charge measured by zeta potential is decreased compared with equivalent polycation-DNA complexes in each case. Atomic force microscopy shows that pHPMA-pTMAEM/DNA complexes are discrete spheres similar to those formed between DNA and simple polycations, whereas pEG-pLL/DNA complexes adopt an extended structure. Biological properties depend on the charge ratio of formation. At optimal charge ratio, pEG-pLL/DNA complexes show efficient transfection of 293 cells in vitro, while pHPMA-pTMAEM/DNA complexes are more inert. Both block co-polymer-DNA complexes show only limited cytotoxicity. Careful selection of block co-polymer structure can influence the physicochemical and biological properties of the complexes and should permit design of materials for specific applications, including targeted delivery of genes in vivo.

Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Key issues in non-viral gene delivery.

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.

Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors.

A major factor limiting the development of non-viral gene delivery systems is the poor characterisation of polyelectrolyte complexes formed between cationic polymers and DNA. The present study uses the fluorescamine reagent to improve characterisation of poly(L-lysine) (pLL)/DNA complexes post-modified with a multivalent hydrophilic polymer by determining the availability of free amino groups. The results show that the fluorescamine reagent can be used to monitor the self-assembly reaction between pLL and DNA and the degree of surface modification of the resultant complexes with a hydrophilic polymer. This experimental approach should enable the preparation of fully defined complexes whose properties can be better related to their biological activity.

Influence of molecular weight on passive tumour accumulation of a soluble macromolecular drug carrier.

The molecular weight-dependence of tumour capture of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers (fractions of mw 22,000-778,000) was studied in vivo using subcutaneous (s.c.) Sarcoma 180 or B16F10 melanoma models. At 10 min, all fractions were already detectable in the tumour (1.5-3% of dose administered per gram) and those of molecular weight greater than the renal threshold showed progressive tumour accumulation up to 20% of dose administered per gram after 72 h in the Sarcoma 180 model. Tumour-selective uptake was confirmed for all copolymer fractions in both tumour models and in the sarcoma 180 model, the ratio (accumulation index, AI) of the AUC in tumour to AUC in skeletal muscle (a typical normal tissue) increasing from six to 12 with increasing copolymer molecular weight. The tumour/blood AI was greater (1-3) in the Sarcoma 180 model than the B16F10 melanoma model (0.4-1.0).

Passive tumor targeting of soluble macromolecules and drug conjugates.

The biodistribution of soluble macromolecules is governed extensively by their ability to penetrate endothelial layers. Many solid tumors possess vasculature that is hyperpermeable to macromolecules, not always correlating with the presence of interendothelial cell fenestrations. The exact physiological mechanisms responsible for this nonspecific leakiness are not yet fully understood. Together with enhanced vascular permeability, however, tumors usually lack effective lymphatic drainage; consequently, they selectively accumulate circulating macromolecules (up to 10% of an i.v. dose per gram in mice). This "enhanced permeability and retention effect" (EPR effect) has been studied extensively, and it is thought to constitute the mechanism of action of SMANCS (styrene-maleic/anhydride-neocarzinostatin), now in regular clinical use in Japan for the treatment of hepatoma. It seems likely that EPR also contributes to the anticancer activity of the N-(2-hydroxypropyl)methacrylamide copolymer-anthracycline conjugates which are shortly to undergo clinical evaluation in the U.K.

The pharmacokinetics of polymer-bound adriamycin.

Adriamycin (ADR) covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via biodegradable (Gly-Phe-Leu-Gly) oligopeptide sequences shows antitumour activity against model tumours in vivo. In this study we have examined the distribution of ADR bound to such HPMA copolymers following intravenous administration to mice (ADR concentration 5 mg/kg). An established fluorimetric HPLC method was used to measure levels of free ADR in plasma and tissue samples, and a new technique was developed to quantitate levels of polymer-bound anthracycline. The high initial levels of free ADR in plasma observed following administration of free drug were absent in the case of polymer-bound ADR, and the subsequently high levels of free ADR seen in other tissues were also abolished. In contrast, the circulating half-life of HPMA copolymer-ADR was approximately 15 times longer than that of the free drug. The initial peak level of free ADR in the heart was reduced 100-fold following administration of drug-conjugate. These alterations in pharmacokinetics may account for the decreased toxicity and improved efficacy reported previously.

A novel dosage approach for evaluation of SMANCS [poly-(styrene-co-maleyl-half-n-butylate) - neocarzinostatin] in the treatment of primary hepatocellular carcinoma.

We report a Phase I/II clinical trial of poly-(styrene-co-maleyl-half-n-butylate)-neocarzinostatin (SMANCS) for intra-arterial treatment of hepatoma. Early patients received 4 or 8 mg SMANCS dissolved in Lipiodol; later patients were treated according to tumour size and degree of filling achieved. SMANCS/Lipiodol drained rapidly from normal liver but was retained within tumour interstitium. Tumour nodules filled with SMANCS/Lipiodol usually stabilised and often regressed. No UICC criteria-defined responses were achieved, partly due to difficulties of filling several lesions simultaneously. Signs of therapeutic activity suggest a more extensive clinical study is warranted.

Harnessing nuclear localization pathways for transgene delivery.

Inefficient transport of DNA from the cytoplasm into the nucleus remains a limiting step in the development of non-viral gene delivery systems. This is particularly acute in non-dividing cells, where entry to the nucleus is thought to occur only through the nuclear pore complex. Active import of physiological proteins is mediated by nuclear localization sequences (NLSs) within cargo proteins such as transcription factors. Here we review current knowledge of this import machinery and consider its exploitation by mammalian viruses. Significant research effort has been directed at incorporating NLSs into synthetic gene delivery systems to take advantage of this physiological pathway. Both non-covalent and covalent methods of conjugation are evaluated, with NLS linkage to both DNA and carrier, and compared with activities of simple cationic polymers. Finally, progress in the field of DNA sequence-specific nuclear import is examined and the current state of the technology assessed.

Gene therapy for colorectal cancer.

Colorectal cancer is an important public health problem worldwide. Gene therapy has therapeutic potential for patients with advanced or recurrent colorectal cancer, incurable by conventional treatments. To date, many strategies of gene therapy have been explored, including mutant gene correction, prodrug activation, immune stimulation and genetically-modified oncolytic viruses. Although the preclinical results of gene therapy for colorectal cancer have shown promise, gene therapy is still at an early stage of clinical development and has not yet shown a significant therapeutic benefit for patients. The main obstacles for introduction of gene therapy to patients are poor targeting selectivity of the vectors and inefficient gene transfer. As the science supporting tumour-selective vectors evolves, gene therapy may expand rapidly in the clinical practice of colorectal cancer treatment.

Reduced cardiotoxicity of doxorubicin given in the form of N-(2-hydroxypropyl)methacrylamide conjugates: and experimental study in the rat.

A rat model was used to evaluate the general acute toxicity and the late cardiotoxicity of 4 mg/kg doxorubicin (DOX) given either as free drug or in the form of three N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. In these HPMA copolymers, DOX was covalently bound via peptide linkages that were either non-biodegradable (Gly-Gly) or degradable by lysosomal proteinases (Gly-Phe-Leu-Gly). In addition, one biodegradable conjugate containing galactosamine was used; this residue was targeted to the liver. Over the first 3 weeks after the i.v. administration of free and polymer-bound DOX, all animals showed a transient reduction in body weight. However, the maximal reduction in body weight seen in animals that received polymer-bound DOX (4 mg/kg) was significantly lower than that observed in those that received free DOX (4 mg/kg) or a mixture of the unmodified parent HPMA copolymer and free DOX (4 mg/kg; P less than 0.01). Throughout the study (20 weeks), deaths related to cardiotoxicity were observed only in animals that received either free DOX or the mixture of HPMA copolymer and free DOX; in these cases, histological investigations revealed marked changes in the heart that were consistent with DOX-induced cardiotoxicity. Sequential measurements of cardiac output in surviving animals that received either free DOX or the mixture of HPMA copolymer and free DOX showed a reduction of approximately 30% in function beginning at the 4th week after drug administration. The heart rate in these animals was approximately 12% lower than that measured in age-matched control rats (P less than 0.05). Animals that were given the HPMA copolymer conjugates containing DOX exhibited no significant change in cardiac output throughout the study (P less than 0.05). In addition, no significant histological change was observed in the heart of animals that received DOX in the form of HPMA copolymer conjugates and were killed at the end of the study. However, these animals had shown a significant increase in heart rate beginning at 8 weeks after drug administration (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Macromolecular derivatives of N,N-di-(2-chloroethyl)-4-phenylene diamine mustard. 2. In vitro cytotoxicity and in vivo anticancer efficacy.

Prodrugs of N,N-di-(2-chloroethyl)-4-phenylene diamine (PDM) based on soluble poly[N5-(2-hydroxyethyl)-l-glutamine] (PHEG) have been evaluated as tumour-targeted drugs. These materials are designed to exploit the enhanced permeability of tumour vasculature, combining a passive tumour tropism with systemic liberation of free PDM. Modification of PDM by coupling via oligopeptide spacers onto a polymeric carrier significantly reduced its cytotoxicity towards different cell types in vitro. On the other hand, incubation of the cells with the PHEG-Gly-Phe-Ala-Leu-PDM conjugate in the presence of collagenase IV led to the release of lethal amounts of free drug, resulting in higher cytotoxicity for this derivative. The PHEG-Gly-Phe-Ala-Leu-PDM conjugate, which is rapidly degraded by lysosomal and tumour-associated enzymes also showed a decreased systemic toxicity in vivo and could be administered at a dose of 8 mg PDM/kg body weight intravenously, compared with just 2 mg/kg for free PDM. Furthermore, this derivative also showed better antitumour activity against a C26 colorectal carcinoma tumour model, compared with no activity for the free drug. The results indicate that the PHEG-Gly-Phe-Ala-Leu-PDM conjugate is a promising candidate for cancer treatment.

DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers.

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4 x 10(-5) g/ml and (3) both components should be mixed together in deionized water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the zeta-potential which finally resulted in formation of a "protein layer" and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the transfection efficiency of DNA complexes with copolymers was always higher than that of the cationic homopolymer slightly increasing with increasing content of HPMA in the copolymers but being about 10-100-times lower than the complexes DNA-poly(L-lysine. In the cytoplasmic injections, it was observed that DNA complexes produced greater gene expression than a direct microinjection of free DNA. The block copolymer complexes were also found to be more efficient than the corresponding simple polycation complexes. In the nuclear microinjection, precisely the opposite behaviour was observed.