Relationship between tumour resistance and the in vitro and in vivo cytotoxicity elicited by Salmonella antigens in immunized mice

Immunization of mice with the attenuated 11RX strain of Salmonella enteritidis (11RX) induces resistance to intraperitoneal (i.p.) tumour growth. Tumour resistance is much greater and lasts for a longer time following i.p. immunization than following intravenous (i.v.) immunization. This paper extends our previous observations that, after this resistance is lost, it can be recalled by a T cell-mediated reaction to an antigenic extract of the bacteria (11RX antigen) which is not protective in unimmunized mice. The duration of this sensitization to 11RX antigen was determined in mice immunized i.p. or i.v. with live 11RX by challenging them at various times after immunization with 131I (or 125I)-labelled Ehrlich ascites tumour (EAT) cells alone or mixed with 11RX antigen. In vivo killing of EAT cells was assayed by monitoring whole-body retention of radioactivity and this was correlated in the same mice with suppression of tumour growth and survival of the mice. The resistance recalled by 11RX antigen was short-lived and in vivo cytotoxic activity had subsided by 6 days after antigen injection. 11RX antigen also recalled the ability of the peritoneal cells to lyse 51Cr-labelled EAT cells in vitro and a close correlation was found between this activity and the cytotoxicity measured in vivo. The adherence properties of the cytotoxic cells and their inhibition by trypan blue indicated that they were macrophages.     

Biodistribution and tumour localisation of a daunomycin-monoclonal antibody conjugate in nude mice with human tumour xenografts

Summary The blood kinetics and tissue distribution of a conjugate of daunomycin and a monoclonal antibody (791T/36) have been examined in mice, including nude mice with human tumour xenografts reactive with the antibody. For this the antibody moiety of the conjugate was labelled with ¹²⁵I and the drug moiety assayed by radioimmunoassay. After radioiodination, the preparation had an immunoreactive fraction in isotopic binding tests with 791T cells of 74%. Both drug and antibody moieties were precipitable with anti-mouse Ig anti-serum. Following i.v. injection, blood clearance of the two components of the conjugate was essentially identical, and with the serumborne conjugate both radiolabel and drug were co-precipitable. In mice with 791T xenografts, the tumour showed localisation of both drug and antibody moieties and at the time of analysis (3 days) tumour levels of drug were over 100 times those seen with free drug. In parallel studies with mice with antigen negative xenografts, there was no preferential localisation of antibody or drug moieties of the conjugate. These studies have shown in vivo stability of this conjugate, and site-specific targetting of an anti-tumour anthracycline.     

Synergistic in vitro and in vivo anti-tumor effect of daunomycin-anti-96-kDa melanoma-associated antigen monoclonal antibody CL 207 conjugate and recombinant IFN-gamma

The mAb CL 207 recognizes a 96-kDa melanoma-associated Ag. The latter is not modulated by antibody but is highly susceptible to induction by immune IFN-gamma on human carcinoma and melanoma cells. The mAb CL 207 does not mediate C- and cell-dependent lysis of human carcinoma and melanoma cells. Conjugation of daunomycin with mAb CL 207 causes a slight reduction of its immunoreactivity and affinity but does not affect its serologic specificity or the toxicity of daunomycin. In combination with IFN-gamma, the daunomycin-mAb CL 207 conjugate displays a selective in vitro and in vivo toxic effect on tumor cells that express the 96-kDa MAA. The synergistic effect of IFN-gamma and daunomycin-mAb CL 207 conjugate is specific. The in vivo toxicity is influenced by the interval between injection of human tumor cells into nude mice and that of IFN-gamma and daunomycin-mAb CL 207 conjugate. The present results suggest that the 96-kDa melanoma-associated Ag may be a useful model to investigate the combined use of IFN-gamma and mAb for the selective destruction of tumor cells.     

Mesothelin enhances invasion of ovarian cancer by inducing MMP-7 through MAPK/ERK and JNK pathways

Ovarian cancer has one of the highest mortalities in malignancies in women, but little is known of its tumour progression properties and there is still no effective molecule that can monitor its growth or therapeutic responses. MSLN (mesothelin), a secreted protein that is overexpressed in ovarian cancer tissues with a poor clinical outcome, has been previously identified to activate PI3K (phosphoinositide 3-kinase)/Akt signalling and inhibit paclitaxel-induced apoptosis. The present study investigates the correlation between MSLN and MMP (matrix metalloproteinase)-7 in the progression of ovarian cancer, and the mechanism of MSLN in enhancing ovarian cancer invasion. The expression of MSLN correlated well with MMP-7 expression in human ovarian cancer tissues. Overexpressing MSLN or ovarian cancer cells treated with MSLN showed enhanced migration and invasion of cancer cells through the induction of MMP-7. MSLN regulated the expression of MMP-7 through the ERK (extracellular-signal-regulated kinase) 1/2, Akt and JNK (c-Jun N-terminal kinase) pathways. The expression of MMP-7 and the migrating ability of MSLN-treated ovarian cancer cells were suppressed by ERK1/2- or JNK-specific inhibitors, or a decoy AP-1 (activator protein 1) oligonucleotide in in vitro experiments, whereas in vivo animal experiments also demonstrated that mice treated with MAPK (mitogen-activated protein kinase)/ERK- or JNK-specific inhibitors could decrease intratumour MMP-7 expression, delay tumour growth and extend the survival of the mice. In conclusion, MSLN enhances ovarian cancer invasion by MMP-7 expression through the MAPK/ERK and JNK signal transduction pathways. Blocking the MSLN-related pathway could be a potential strategy for inhibiting the growth of ovarian cancer.     

Effective drug-antibody targeting using a novel monoclonal antibody against the proliferative compartment of mammalian squamous carcinomas

Summary mAb 174H.64, which selectively recognizes an epitope expressed on the proliferating cells of mammalian squamous carcinomas, was covalently coupled to daunomycin (DM) by an acid-sensitive linker and tested for its selective cytotoxicity for squamous carcinomas. A murine lung squamous carcinoma model for chemoimmunotherapy using mAb 174H.64-DM conjugates was developed. This model utilizes the KLN-205 cell line, which metastasizes to the lungs following i.v. injection and shows a pattern of growth similar to those of spontaneous squamous carcinomas, characterized by highly proliferative cells at the periphery of the tumor (reactive with 174H.64) with the keratinized differentiated cells toward the center (not reactive with 174H.64). 174H.64-DM conjugates showed marked and specific cytotoxicity against KLN-205 cells both in vitro and following i.v. injection of the immunoconjugate in mice with established lung metastases. The conjugate was nearly as effective as daunomycin alone when incubated in vitro with KLN-205 cells and much more effective than daunomycin alone in vivo or other control immunoconjugates, which were ineffective. Finally, while the free 174H.64 mAb produced a significantly increased time of survival of mice bearing KLN-205 metastases, a much greater survival was found with mice treated with the 174H.64-DM immunoconjugate, some mice apparently demonstrating long-term survival (>100 days). We conclude that mAb 174H.64 may have potential therapeutic benefit against squamous carcinoma.This work was supported by the Medical Research Council of Canada and Biomira Inc.     

Lumiflavin increases the sensitivity of ovarian cancer stem‐like cells to cisplatin by interfering with riboflavin

Here, we used lumiflavin, an inhibitor of riboflavin, as a new potential therapeutic chemosensitizer to ovarian cancer stem‐like cells (CSCs). This study demonstrates that the enrichment of riboflavin in CSCs is an important cause of its resistance to chemotherapy. Lumiflavin can effectively reduce the riboflavin enrichment in CSCs and sensitize the effect of cisplatin Diamminedichloroplatinum (DDP) on CSCs. In this study, CSCs of human ovarian cancer cell lines HO8910 were separated using a magnetic bead (CD133+). We also show the overexpression of the mRNA and protein of riboflavin transporter 2 and the high content of riboflavin in CSCs compared to non‐CSCs (NON‐CSCs). Moreover, CSCs were less sensitive to DDP than NON‐CSCs, whereas, the synergistic effect of lumiflavin and DDP on CSCs was more sensitive than NON‐CSCs. Further research showed that lumiflavin had synergistic effects with DDP on CSCs in increasing mitochondrial function damage and apoptosis rates and decreasing clonic function. In addition, we found that the combination of DDP and lumiflavin therapy in vivo has a synergistic cytotoxic effect on an ovarian cancer nude mice model by enhancing the DNA‐damage response and increasing the apoptotic protein expression. Notably, the effect of lumiflavin is associated with reduced riboflavin concentration, and riboflavin could reverse the effect of DDP in vitro and in vivo. Accordingly, we conclude that lumiflavin interfered with the riboflavin metabolic pathways, resulting in a significant increase in tumour sensitivity to DDP therapy. Our study suggests that lumiflavin may be a novel treatment alternative for ovarian cancer and its recurrence.     

Local interleukin 2 therapy is most effective against cancer when injected intratumourally

Local interleukin 2 (IL-2) therapy is more effective against systemic tumours than systemic IL-2 therapy, but it remains unclear whether IL-2 should be injected intratumourally or peritumourally. To investigate this question, we treated DBA/2 mice bearing a large subcutaneous syngeneic SL2 lymphoma with either intra or peritumoural IL-2 therapy. Both applications enhanced survival, but intratumourally injected IL-2 was more effective than peritumourally injected IL-2. Tumours started to regress 4 days after IL-2 injection. Tumour cells died at the IL-2 injection site, although IL-2 is not directly cytotoxic for SL2 cells in vitro. Tumour cell death correlated well with oedema and extravascular erythrocytes, but less with leukocyte infiltrates. In mice bearing two s.c. tumours, intratumoural application therapy of IL-2 in one tumour caused decrease in size of both tumours in 4–9 days after therapy. However, the IL-2 treated tumours regressed more strongly than the untreated tumours. We conclude that vascular leakage and/or tissue destruction inside the tumour may contribute to the enhanced effect of intratumoural IL-2 therapy compared to peritumoural IL-2 therapy. Hence, we recommend applying of intratumoural rather than peritumoural IL-2 therapy.     

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours

 Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL. Received: 20 June 1996 / Accepted: 4 January 1997     

A double fluorescence method for determining doxorubicin distribution and vascular supply in the mouse kidney

Freeze-sectioning followed by freeze-drying completely preserves the topographic distribution of soluble substances, with the additional advantage of permitting permanent mounting. This rapid and effective technique was applied to study the distribution of fluorescence of the cytotoxic drug Doxorubicin in microscopic slices from in vivo treated animals. A fluorescent conjugate of different fluorescence, Blankophor-G-Dextran, which remained in the vessels, was used in the same living animals to characterize the vascular supply in the kidney of treated mice in relation to Doxorubicin distribution.     

Antitumor activity of a thioether-linked immunotoxin: OVB3-PE

A thioether-linked immunotoxin was made between Pseudomonas exotoxin and the monoclonal antibody OVB3. This conjugate, OVB3-PE, was cytotoxic for the human ovarium cancer cell line OVCAR-3 (ID of 2.5 x 10(-12) M) and it was therefore tested for antitumor activity in a nude mouse model of ovarian cancer. This model employs the injection of a lethal number of OVCAR-3 cells into the peritoneal cavity of nude mice. When 0.2-1 micrograms of OVB3-PE was injected intraperitoneally on three successive days beginning 3-5 days after OVCAR-3 cell implantation, the survival of the tumor-bearing mice was increased 2-4-fold compared to that of untreated control mice. Median survival times for control mice ranged from 44 to 50 days while survival times of 150 days or greater were seen in mice treated with OVB3-PE. When OVB3-PE administration was delayed until 2-4 weeks after tumor cell implantation, OVB3-PE treatment also showed antitumor activity, but the duration of survival was less than with the early treatments. OVB3-PE was also cytotoxic for MCF-7 breast carcinoma cells, HT-29 colon carcinoma cells, and A431 epidermoid carcinoma cells.     

Treatment with HPMA copolymer-based doxorubicin conjugate containing human immunoglobulin induces long-lasting systemic anti-tumour immunity in mice

Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox–PHPMA–HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10–20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8⁺ lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.     

In vivo and in vitro characterisation of a protoporphyrin IX-cyclic RGD peptide conjugate for use in photodynamic therapy.

Increasing treatment specificity is one of the major aims of cancer research. Photodynamic therapy is a clinically proven treatment for some cancers and certain other diseases. Photosensitisers generally have little intrinsic selectivity for tumours and any accumulation is dependent upon the type of tumour involved. Increasing tumour selective accumulation could improve the efficacy of PDT and reduce any risk of side effects caused by photosensitiser accumulation in non-target tissue. In order to target photosensitisers to tumours, a cyclic peptide, cRGDfK (arginine-glycine-aspartic acid-phenylalanine-lysine) has been synthesised using solid phase peptide chemistry and conjugated to the porphyrin photosensitiser, protoporphyrin IX. The arginine-glycine-aspartic acid (RGD) motif has been shown to specifically bind alphavbeta3 integrins, heterodimeric glycoproteins upregulated on the surface of proliferating endothelial cells such as those in tumour neovasculature. This study reports the synthesis, in vitro and in vivo characterisation of this novel compound and compares its properties to the free photosensitiser. The individual components in our system, protoporphyrin IX and cRGDfK retain their respective photodynamic and integrin binding activity following the coupling step and produce a conjugate of high purity. The PpIX:cRGDfK conjugate is shown to be a good photosensitiser in vitro in the integrin positive human SiHa cell line and in vivo in a mouse CaNT tumour model. Moreover, pharmacokinetic analysis of PpIX:cRGDfK treated mice shows significant retention and accumulation of photosensitiser in tumour tissue with higher tumour : normal tissue ratios than the free photosensitiser. However, although the conjugate shows this higher accumulation and improved tumour : non-target tissue ratios, the overall in vivo PDT effect, between dose-light intervals of 0 and 6 h, is not significantly better than for free protoporphyrin IX This is possibly due to differences in the target environment or in the subcellular localisation of the compounds.     

Berberine inhibits the chemotherapy‐induced repopulation by suppressing the arachidonic acid metabolic pathway and phosphorylation of FAK in ovarian cancer

Objectives

Cytotoxic chemotherapy is an effective and traditional treatment of ovarian cancer. However, chemotherapy‐induced apoptosis may also trigger and ultimately accelerate the repopulation of the small number of adjacent surviving cells. This study mainly focused on the tumour cell repopulation caused by chemotherapy in ovarian cancer and the adjunctive/synergistic effect of Berberine on the prevention of tumour repopulation.

Materials and methods

The transwell system was used to mimic the co‐culture of surviving ovarian cancer cells in the microenvironment of cytotoxic chemotherapy‐treated dying cells. Tumour cell proliferation was observed by crystal violet staining. AA and PGE₂ levels were measured by ELISA, and changes of protein expression were analysed by Western blot.

Results

Chemotherapy drug VP16 treatment triggered AA pathway, leading to the elevated PGE₂ level, and ultimately enhanced the repopulation of ovarian cancer cells. Berberine can block the caspase 3‐iPLA₂‐AA‐COX‐2‐PGE₂ pathway by inhibiting the expression of iPLA₂ and COX‐2. Berberine can also reverse the increased phosphorylation of FAK caused by abnormal PGE₂ level and thus reverse the repopulation of ovarian cancer cells after VP16 treatment.

Conclusions

Our observation suggested that Berberine could inhibit the chemotherapy‐induced repopulation of ovarian cancer cells by suppressing the AA pathway and phosphorylation of FAK. And these findings implicated a novel combined use of Berberine and chemotherapeutics, which might prevent ovarian cancer recurrence by abrogating early tumour repopulation.

     

Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient

Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.     

Photo-immunotargeting with haematoporphyrin conjugates activated by a low-power He-Ne laser

Summary A combined method has been developed for selective cytolysis in vitro as well as in vivo using a photosensitizer haematoporphyrin-protein conjugate as the targeting molecule and low-power He-Ne laser (632.8 nm) irradiation in order to activate the sensitizer to its excited, toxic triplet energy state. The specificity of the procedure was demonstrated in vitro by purging a mixed cell population from one component, and in vivo in an animal (nude mice) xenograft tumour model, where human cancer cells were destroyed by the immunotargeting method using monoclonal-antibody-haematoporphyrin (mAb-HP) conjugate (a-PNAr-I mAbs, which bind to the cell surface antigens of gastric cancer cells) and soft laser irradiation. The cell destruction was dependent on the doses of both mAb-HP and He-Ne laser light energy, and occurred only in target cell populations.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.     

Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype

Tumor-associated macrophages (TAM) may have tumor-promoting activity, but it is not clear how their phenotype is achieved. In this study, we demonstrate that ovarian cancer cells switch cocultured macrophages to a phenotype similar to that found in ovarian tumors. Tumor cells caused dynamic changes in macrophage cytokine, chemokine, and matrix metalloprotease mRNA, and protein-inducing mediators that are found in human cancer. Macrophage mannose, mannose receptor, and scavenger receptors (SR-As) were also up-regulated by coculture, but not by conditioned medium. To further validate the model, we studied SR-A regulation on TAM in vitro and in vivo. Coculture of murine macrophages from mice deficient in TNF-alpha or its receptors revealed that TNF-alpha was key to SR-A induction via its p75 receptor. SR-A expression was also reduced in TAM from ovarian cancers treated with anti-TNF-alpha Abs or grown in TNF-alpha(-/-) mice. Chemical communication between tumor cells and macrophages may be important in regulating the cancer cytokine microenvironment.     

<Emphasis Type="Italic">In Vivo</Emphasis> Anticancer Efficacy and Toxicity Studies of a Novel Polymer Conjugate <Emphasis Type="Italic">N</Emphasis>-Acetyl Glucosamine (NAG)–PEG–Doxorubicin for Targeted Cancer Therapy

A novel polymer–drug conjugate, polyethylene glycol–N-(acetyl)-glucosamine–doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer–drug conjugate comprised of polyethylene glycol–maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.