Nanotechnology for Treating Cancer: Pitfalls and Bridges on the Path to Nanomedicines

June 8, 2015, by Rachael Crist and Scott McNeil

Editor's note: For more than 30 years cancers caused by oncogenic RAS mutants have resisted direct therapeutic attack.  Using nanomedicines to target RAS-driven cancers at the genomic or RNA level while sparing normal tissues has engaged the imagination of researchers, yet these aspirations have not yet yielded any drugs used in human patients.  As described below, the Nanotechnology Characterization Laboratory (NCL) of the NCI has helped universities, companies, and institutes evaluate hundreds of nanomedicine candidates.  Rachael Crist and Scott McNeil of the NCL summarize the challenges that must be overcome on the path to successful nanomedicines, including those that may someday target mutant RAS.

Nanotechnology is often touted as one of the most promising drug delivery innovations in today’s fight against cancer. Typically not drugs themselves, nanoparticles have the potential to deliver traditional cancer drugs to tumors with fewer side effects, or to enable non-traditional drugs (e.g., proteins or nucleic acids) to be targeted to kill cancer cells.  However, since the mid-nineties only two nanoparticle cancer treatments have been approved by the FDA. Doxil (Janssen Biotech), a liposomal formulation of doxorubicin, was the first nanomedicine approved (1995). In 2005 the albumin-bound paclitaxel formulation Abraxane (Celgene Corp.) was approved, largely by virtue of reduced side effects in the treatment of solid tumors. The question naturally arises, why aren’t there more approved nanodrugs on the market? Why is their development taking so long? Based on our experiences the lag between inception and delivery in the development of nanotechnology-based therapeutics can be ascribed to the complexity of the nanomaterials themselves. Nanomaterials are not pure entities. On the contrary, they are complex, polydisperse, and oftentimes multifunctional formulations—the term in the field is "non-biological complex drugs" (NBCDs). Many different physical and chemical traits must be fine-tuned for each application, including nanoparticle size, charge, surface chemistry, and hydrophobicity, and this tuning process requires a suite of skills and technologies that often must be developed iteratively.  "One size does not fit all" is a truism in the field of nanomedicine.

For the past ten years the Nanotechnology Characterization Lab (NCL) of the National Cancer Institute has provided comprehensive nanomaterial characterization, testing and evaluation services to nanomedicine developers around the world. The NCL has analyzed over 300 different nanoformulations and worked with nearly 100 academic, government, and commercial organizations, affording a unique opportunity to study the advantages and disadvantages of various platforms, drugs, surface coatings, targeting ligands, etc. A summary of our experience with many of the most common pitfalls encountered by nanomedicine developers was recently published.¹

With nanoparticle-enabled delivery of chemotherapeutics there is an opportunity to deliver more of the cytotoxic drug to the intended site, offering reduced off-target toxicities and / or enhanced efficacy (because the "maximum tolerated dose" can be higher). Formulation using nanotechnology can alter biodistribution, clearance, and pharmacokinetics compared to the legacy drug. Employing what is known as the enhanced permeability and retention (EPR) effect, nanoparticles can accumulate in tumor tissues because of their leaky vasculature and compromised lymphatic drainage.² To take advantage of the EPR effect however, nanoparticles must be precisely engineered to evade the mononuclear phagocyte system (MPS). For example, nanomedicines should be small enough to avoid uptake by the liver and spleen (<250 nm), yet large enough to avoid clearance by the kidneys (>10 nm), and should remain in circulation long enough to allow for significant tumor accumulation (t1/2 > 3 hr).  A nanomaterial’s physicochemical properties are known to influence its biological performance. For example, a size difference as little as 2 nm has been shown to alter the route of clearance, and surface modification has been shown to alter biodistribution.^3-5

It goes without saying that the most critical step in nanomedicine evaluation is thorough and well-documented characterization of each batch of material. Without a comprehensive understanding of the formulation its biological analysis can be easily misinterpreted. Starting materials should be characterized; intermediate materials should be characterized and archived for side-by-side biological evaluation (e.g. non-targeted vs. targeted nanomaterials); and of course, the final material should be characterized. It is imperative to ascertain which parameters are the most important to measure, which ones affect biological activity, and what assay techniques are most appropriate. A short list of essential parameters to investigate includes 1) the level of polydispersity that is acceptable to maintain the desired efficacy; 2) whether or not the surface coatings/targeting ligands are covalently attached or simply physisorbed; 3) the level of surface coverage required for optimal biological performance; 4) and the stability (temperature, pH, shelf-life, in biological matrix, etc.) of the formulation. There are a number of excellent articles that address the importance of these and other characterization aspects.^{3, 6-9}

Additional challenges arise when delivering agents such as RNA, DNA, or proteins, especially avoiding undesired immune responses. Although many nanoparticles without these agents can also elicit immunological reactions, nanoparticles containing oligos or proteins are particularly susceptible. Some commonly observed toxicities include anemia, neutropenia, thrombosis, complement activation, cytokine induction, and allergic reactions. Screening for this variety of potential adverse conditions can be a challenge, and it is often difficult to predict which assays will be most relevant for a given formulation. The NCL has more than a dozen in vitro immunological assays tailored for nanomedicine evaluation. These are available for download at http://ncl.cancer.gov for interested researchers.

Sterility and endotoxin contamination are other aspects many researchers fail to address in the developmental process. Almost one-third of the materials submitted to the NCL have endotoxin levels exceeding US FDA limits. Even though materials submitted to the NCL are at the preclinical stage, high levels of endotoxin can interfere with the correct interpretation of many assays. Without a proper screen for endotoxin, toxicity may be mistakenly assigned to the nanomaterial, when in fact it stems from contamination during the synthesis and/or purification process. The NCL has studied endotoxin quantification in nanomaterials extensively and has several manuscripts offering more elaboration on the trials and tribulations of this topic.^10-12

Generic versions of nanomedicines, or nanosimilars, pose even more challenges. Recently the FDA approved the first “generic” nanomedicine. A generic version of Doxil (Sun Pharma) was approved for the treatment of ovarian cancer in 2013, nearly 20 years after approval of the innovator product. Researchers are now beginning to develop new and improved methods to verify the bioequivalence of these follow-on nanomedicines. However, there is an inherent challenge in defining similarity for a formulation that is naturally polydisperse, and determining whether or not these differences are meaningful. Because of the novelty of this area, the potential regulatory hurdles and additional characterization requirements for nanosimilars may not yet be fully realized.

Nanomedicine research is not unrealistic or unattainable. There are many who do this work superbly. But for the novice nano-researcher, here are a few key recommendations:

• Screen for endotoxin early.
• Ensure the biocompatibility of all formulation components.
• Monitor the complete removal of potentially toxic excipients and side products from the manufacturing and purification process.
• Certify batch-to-batch consistency and define acceptance criteria.
• Assess nanoparticle stability and drug release rates in vivo or in an appropriate biological matrix.
• Don’t ignore immunological assessments.
• Don’t skimp on proper controls and comparisons for in vivo studies. While this may save on costs in the short term, it usually doesn’t in the long term.  
• It is never too early to think about the big picture and how the research product should ultimately materialize. Researchers who focus on achieving a single optimal preclinical result often lose sight of clinical and regulatory requirements and end up facing unexpected (i.e., time consuming and costly) hurdles in their development path.

A variety of NCL services are available to researchers pursuing nanomedicine translation. Researchers developing cancer nanomedicines are eligible to apply for the free NCL Assay Cascade testing services. Formulations are accepted via a quarterly application process; more information about this service, as well as acceptance and evaluation criteria can be found on the NCL’s website. Other services are also available via a Collaborative Research and Development Agreement (CRADA). These include characterization of non-oncology based nanomedicines, nanotechnology reformulation services, and more. Submitting investigators must provide funding for services under the cCRADA, but NCL only charges for direct costs—there is zero profit associated with these contracts. For more information about the NCL or for questions, please email us at ncl@mail.nih.gov.

References
1. Crist RM, et al. Integr Biol (Camb) 2013; 5: 66-73.
2. Matsumura Y, Maeda H. Cancer Res 1986; 46: 6387-92.
3. Adiseshaiah P, Hall JB, McNeil S. WIREs Nanomed Nanobiotechnol 2010; 2: 99-112.
4. Kobayashi H,Brechbiel MW. Molecular Imaging 2003; 2: 1-10.
5. Paciotti GF, et al. Drug Delivery 2004; 11: 169-83.
6. Clogston JD, Patri AK. Handbook of Immunological Properties of Engineered Nanomaterials, Dobrovolskaia MA, McNeil SE, Eds. World Scientific Publishing: 2013; pp 25-52.
7. Malik N, et al. J. Controlled Release 2000; 65: 133-48.
8. Salvador-Morales C, et al. Biomaterials 2009; 30: 2231-40.
9. Aggarwal P, et al. Adv Drug Delivery Rev 2009; 61: 428-437.
10. Dobrovolskaia MA, McNeil SE. In Handbook of Immunological Properties of Engineered Nanomaterials, Dobrovolskaia MA, McNeil SE, Eds. World Scientific Publishing: 2013; pp 77-115.
11. Dobrovolskaia MA, et al. Nanomedicine (Lond) 2010; 5: 555-562.
12. Dobrovolskaia MA, et al. Nanomedicine (Lond) 2014, 9: 1847-56.

Scott McNeil is the Director of the Nanotechnology Characterization Laboratory at the Frederick National Laboratory for Cancer Research. Rachael Crist is a Scientist in the NCL, providing project management and technical writing support.