Oral cancer is one of the most common and lethal cancers in the world. Combination chemotherapy coupledwith nanoparticle drug delivery holds substantial promise in cancer therapy. This study aimed to evaluate theefficacy and safety of two dosages of our novel pH and temperature sensitive doxorubicin-methotrexate-loadednanoparticles (DOX-MTX NPs) with attention to the MMP-2 mRNA profile in a 4-nitroquinoline-1-oxide inducedoral squamous cell carcinoma (OSCC) model in the rat. Our results showed that both IV and oral dosages ofDOX-MTX NP caused significant decrease in mRNA levels of MMP-2 compared to the untreated group (p<0.003).Surprisingly, MMP-2 mRNA was not affected in DOX treated compared to cancer group (p>0.05). Our resultsindicated that IV dosage of MTX-DOX is more effective than free DOX (12 fold) in inhibiting the activity ofMMP-2 in OSCCs (P<0.001). Furthermore, MMP-2 mRNA expression in the DOX-MTX treated group showeda significant relation with histopathological changes (P=0.011). Compared to the untreated cancer group, weobserved no pathological changes and neither a significant alteration in MMP-2 amount in either of healthycontrols that were treated with oral and IV dosages of DOX-MTX NPs whilst cancer group showed a high levelof MMP-2 expression compared to healthy controls (p<0.001).Taking together our results indicate that DOXMTXNPs is a safe chemotherapeutic nanodrug that its oral and IV forms possess potent anti-cancer propertieson aggressive tumors like OSCC, possibly by affecting the expression of genes that drive tumor invasion andmetastasis.