A nanoparticle that can deliver chemotherapy drugs directly to multiple myeloma (MM) cells and overcome cell-adhesion-mediated drug resistance (CAM-DR) is reported by researchers at the University of Notre Dame in Indiana. The micellar nanoparticles carry both doxorubicin and peptide antagonists of very late antigen-4 (VLA-4), a cell surface protein expressed on MM cells that plays a key role in mediating the adhesion of MM cells to bone marrow stroma, which is implicated in the development of CAM-DR.
Basar Bilgiçer, Ph.D., and colleagues say that tests in a mouse xenograft model of MM showed that in comparison with treatment using free doxorubicin, their VLA-4 targeting, doxorubicin-conjugated multifunctional nanoparticles (NPDox/VLA4-pep) preferentially homed in on MM tumors and resulted in dramatic tumor growth inhibition, while preventing CAM-DR and reducing overall system toxicity. The investigators report their development in Nature’s Blood Cancer Journal, in a paper titled “Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo.”
Adhesion of MM cells to bone marrow stroma is a major factor in the development of CAM-DR, and VLA-4 (also known as α4β1 integrin) has been found to play a key role as a receptor in this adhesion process. The Notre Dame researchers thus set out to develop a multifunctional nanoparticle that would be capable of targeting chemotherapy directly to MM cells, but also combine anti-adhesion activity to prevent the development of CAM-DR.
The lipid nanoparticles were synthesized from the lipid-PEG block copolymer DSPE-PEG2000 which, when placed in water self-assembles into micelles. The team then used a synthetic strategy they had devised to conjugate the anti-VLA4-peptides (VLA4-peps) to the nanoparticles, and subsequently attached the doxorubicin molecules via an acid-labile bond. This pH-sensitive bond ensured that the cytotoxic drug would only be released once the nanoparticles were taken up by the MM cells through endocytosis, as the endosomes have an acidic environment.
In order to compare the functions of the resulting NPDox/VLA4-pep nanoparticles, the investigators also generated a set of control nanoparticles that carried only doxorubicin (NPDox), only VLA4-pep (NPVLA4-pep), doxorubicin plus a nonspecific peptide (NPDox/ns), or just a nonspecific-peptide (NPns).
An initial set of cellular uptake studies determined that conjugating 20 VLA4-pep molecules per nanoparticle resulted in the greatest level of receptor-mediated uptake of the nanoparticles by MM cells. Further in vitro studies demonstrated that the optimized NPDox/VLA4-pep nanoparticles were highly cytotoxic to MM cells, and although the cytotoxic effects were slower to become evident when compared with free doxorubicin, by 72 hours after administration the NPDox/VLA4-pep nanoparticles showed a similar level of cytotoxicity to free doxorubicin.
The researchers point out that they expected free doxorubicin to show a faster onset of cytotoxic effects as the drug is taken up by MM cells rapidly via passive diffusion, whereas the NPDox/VLA4-pep nanoparticles have to be internalized by the cells via endocytosis before the doxorubicin is released. In comparison with the high levels of cytotoxicity demonstrated by the NPDox/VLA4-pep nanoparticles, the control NPDox nanoparticles showed far less cytotoxicity at 48 and 72 hours, supporting VLA-4’s role in nanoparticle uptake, the authors point out.
In vitro assays confirmed that NPVLA4-pep nanoparticles (i.e., without conjugated doxorubicin so as not to kill the test cells) inhibited adhesion of NCI-H929 MM cells to fibronectin in a dose-dependent manner, providing an initial indication that blocking VLA-4 activity might lessen the development fo CAM-DR. In a follow-on set of assays the team compared the effects of either free doxorubicin, or the doxorubicin-carrying NPDox/VLA4-pep nanoparticles on MM cells grown on fibronectin-coated plates to enable adhesion. The results of these tests showed that while administration of free doxorubicin had a much reduced cytotoxic effect due to the CAM-DR caused by adhesion of the MM cells to fibronectin, treatment using NPDox/VLA4-pep resulted in significant cell-killing, indicating that the nanoparticles overcame the CAM-DR.
Essentially, the authors state, while free doxorubicin and NPDox/VLA4-pep nanoparticles were equally as cytotoxic on MM cells in suspension, when the cancer cells were cultured in the presence of fibronectin, the NPDox/VLA4-pep nanoparticles had a far more potent anticancer effect than free doxorubicin. “These results establish the significance of targeting MM cells as well as their interactions with the microenvironment in the design of more effective novel therapeutics.”
The researchers finally moved on to test the NPDox/VLA4-pep nanoparticles in vivo, in SCID mice with palpable NCI-H929 tumors. Having previously determined the maximum tolerated dose of the nanoparticles, the team treated four cohorts of tumor-bearing animals using multiple intravenous injections of either free doxorubicin, NPDox/VLA4-pep, NPDox or PBS (as a control). The results showed that while both the free doxorubicin and NPDox/VLA4-pep treatments resulted in dramatic tumor growth inhibition, animals given free doxorubicin lost huge amounts of body weight and demonstrated significant systemic toxicity, so had to be euthanized. In contrast, the NPDox/VLA4-pep-treated cohort lost far less body weight. NPDox therapy did show some anticancer activity, but was far less effective than the NPDox/VLA4-pep at inhibiting tumor growth.
Encouragingly, while the NPDox/VLA4-pep did accumulate in a number of organs tested tested, the nanoparticles accumulated preferentially in the tumors, and reached 10-fold higher intratumoral levels than free doxorubicin, and 5-fold higher levels in tumors than NPDox, the authors stress. This finding supported the notion that incorporating VLA4-pep to the nanoparticles not only facilitates uptake by MM cells and inhibits CAM-DR, but also enhances targeting to MM tumors.
In fact, blood count tests confirmed that the NPDox/VLA4-pep therapy resulted in far less toxicity in terms of white blood cell and thrombocyte counts, and demonstrated far less toxicity with respect to kidney and liver weight, hepatocellular hypertrophy and degeneration, and spleen fibrosis. “Altogether, these results indicate that NPDox/VLA4-pep showed decreased overall systemic toxicity than free doxorubicin,” the researchers state. “We have harnessed nanotechnology to develop a combinational therapy approach for MM, where doxorubicin-conjugated nanoparticles selectively targeted VLA-4 expressing MM cells, prevented development of CAM-DR, and dramatically inhibited tumor growth with overall reduced systemic toxicity. Taken together, this study provides the preclinical rationale for the clinical evaluation of VLA-4 targeting, doxorubicin-conjugated multifunctional nanoparticles to improve patient outcome.”