Drug resistance is a pervasive problem in cancer therapy that results in a vast majority of tumor treatment failure in metastatic cancer cases. Of all of the ND-drug agents that have been synthesized, ND-anthracycline agents are highly scalable and they potently bind the drug compounds that result in marked improvements in efficacy and safety. The ability to rapidly synthesize nanoparticle-tagged drugs that are too large to be effluxed from a tumor mass, coupled with an ability to prevent early systemic release and systemic toxicity and off-target effects would represent a very promising nanomedicine-based route to overcome drug resistance. A recent study demonstrated that NDX is among the most promising platforms for this indication due to the remarkably scalable nature of its synthesis protocol. NDX particles were systemically administered in mice via tail vein injection [27]. Subsequent toxicity measurements of serum IL-6 and serum ALT even at high ND doses indicated no apparent toxicity, demonstrating that the NDs were well tolerated. The impact of NDX injections on myelosuppression was evaluated. NDX administration resulted in no apparent myelosuppression, while the administration of Dox alone resulted in marked reductions in white blood cell count below threshold values. Myelosuppression is the dose-limiting side effect of chemotherapy and can cause major side effects such as superinfections and patient mortality.

With regard to efficacy, unmodified Dox administration at 100 μg equivalence resulted in virtually no efficacy. NDX administration at the same dosage equivalence resulted in a marked decrease in tumor size. When the drug dosage equivalence was doubled, unmodified Dox administration resulted in accelerated animal mortality. When the lethal dosage was delivered via ND, all of the animals survived, and the tumor treatment efficacy was further enhanced, resulting in the smallest tumors observed in the study (Fig. 2). This demonstrated that the ND platform was capable of mediating major improvements to chemotherapeutic tolerance [27].

This initial demonstration of preclinical NDX administration demonstrated that there was no premature drug elution even with a therapeutic that is bound via physisorption. This was confirmed by the fact that premature drug release would have resulted in both apparent myelosuppression due to the circulation of free drug, as well as no therapeutic efficacy since the Dox would be separate from the ND and effluxed. The absence of myelosuppression and marked improvements in efficacy confirmed that potent interaction of NDs with Dox served as an important mediator of both significantly enhanced efficacy and safety, even in a passively targeted system.

The initial validation of NDX systemic administration as an effective strategy for drug-resistant breast and liver tumor therapy served as a promising passively targeted approach. The improved drug tolerance and therapeutic efficacy observed even with the lethal Dox dosage served as a foundation for the continued development of NDX toward clinical use. A recent study has synthesized a variation of the NDX complex in that the Dox compound was replaced with Epi [36]. The disease model in this study was the MDA-MB-231 triple-negative breast cancer (TNBC) that has been shown to overexpress the epidermal growth factor receptor (EGFR) [36]. Therefore, the ND-Epi complexes were encapsulated within liposomes that were surface functionalized with the EGFR antibody (Fig. 3). This architecture was employed in order to maximize drug loading on the ND surface due to the fact that the NDX study was able to confirm that the ND-anthracycline interaction was capable of enhancing intratumoral drug retention to prevent efflux and improve efficacy, circulatory half-life by a factor of 10, and eliminate myelosuppression, among other benefits. These ND-Lipid hybrid particles (NDLPs) thus served as an actively targeted variation of the ND-anthracycline complexes that were previously scalably synthesized [36].

NDLP synthesis was accomplished using a rehydration process of lipid thin films that were comprised of cholesterol and biotinylated lipids and ND solution [36]. By harnessing biotin–streptavidin chemistry, the anti-EGFR antibodies were conjugated to the liposome surface. Characterization of NDLP synthesis was done using a broad range of methodologies including zeta potential analysis and dynamic light scattering (DLS) in order to assess particle size, Fourier Transform Infrared Spectroscopy (FTIR), flow cytometry in order to quantify successful NDLP synthesis and to differentiate the NDLPs from free NDs and lipids, and cryogenic transmission electron microscopy (cryo-TEM) in order to image NDLP formation. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and enzyme-linked immunosorbent assay (ELISA) were used to assess lipid presence and confirm anti-EGFR antibody conjugation, respectively [36].

Comprehensive in vitro assessment of anti-EGFR functionalized NDLP targeting efficacy was performed. Epidermal Growth Factor (EGF) flooding studies resulted in gradually decreasing targeting efficacy as a result of increasing the EGF concentration which was a demonstration of successful anti-EGFR antibody conjugation and targeting abilities. With regard to preclinical efficacy, targeted (anti-EGFR antibody functionalized) and untargeted NDLP complexes, as well as phosphate buffered saline (PBS) and unmodified Epi were utilized as test conditions [36]. Unmodified Epi administration resulted in accelerated animal mortality that was similarly observed with unmodified Dox administration. Untargeted NDLP administration immediately improved drug tolerance as early mortality was no longer observed. Furthermore, tumor growth was clearly delayed. Interestingly, the administration of the anti-EGFR functionalized NDLPs resulted in tumor regression to the point where they were no longer visible, which is a promising finding for actively targeted ND-chemotherapeutic compounds (Fig. 4).

With regard to ND biocompatibility, this study provided among the most comprehensive assessments of ND serum and urine toxicity. A broad range of markers was analyzed including serum ALT, ALK and AST, hematocrit, blood urea nitrogen (BUN), bilirubin, white blood cell differential, hemoglobin, and red blood cell counts, among other readouts. In all cases, ND and NDLP administration resulted in no apparent toxicity that further substantiated the promise of the ND platform for drug delivery.

A key barrier to glioblastoma therapy is the need for the therapeutic to traverse the blood–brain barrier (BBB) [37]. In addition, off-target toxicity is of a major concern because nonspecific cell death in the central nervous system (CNS) results in irreversible damage since CNS neurons do not regenerate. Therefore, localizing drug release to the specific tumor-containing regions, when relevant, is of particular importance.

While the ability for NDX to cross the BBB should be further investigated, a recent study that utilized convection-enhanced delivery (CED), the stereotactic application of a catheter to directly deliver NDX into brain tumors demonstrated improved tumor localization and clear improvements to treatment efficacy in multiple tumor models [38]. Specifically, the C6 rodent model and U251MG, an aggressive human model, were both investigated in a rat model. Initial in vitro studies demonstrated that NDX retention within both cell lines were improved compared to free drug administration. In addition Ki67 and caspase studies demonstrated enhanced cancer cell death mediated by NDX administration compared to free Dox administration. Preclinical retention studies showed that NDX administration in the right lobe of the brain resulted in the presence of Dox at the 72 h time point, while the administration of Dox alone resulted in rapid drug dissipation with no drug presence at 72 h. Importantly, toxicity studies were conducted to examine the presence of edema and swelling in the left lobe of the brain following right lobe administration. While PBS and ND administration in the right lobe resulted in healthy left lobe, unmodified Dox administration into the right lobe resulted in significant swelling in the left lobe, serving as an indicator that free Dox administration could cause major off-target toxicity, even with localized CED administration. It should be noted that NDX administration into the right lobe resulted in healthy left lobe. This was an important finding confirming that Dox could be confined to the tumor-containing regions of the brain via NDX which may markedly improve the tolerance of CED Dox injection (Fig. 5).

Extensive preclinical studies pertaining to NDX efficacy and safety in both C6 and U251MG were conducted. In the U251MG model, tumors observed following PBS and ND administration resulted in animal mortality. Dox administration resulted in an observable reduction in tumor size. However, NDX administration resulted in a significant lengthening of animal survival and cancer cell apoptosis. In the C6 model, the control and Dox-only conditions resulted in largely the same outcomes as those observed in the U251 model. However, NDX administration resulted in tumor regression to the point where the luciferase signal emitted from the tumors were no longer observable and pronounced improvements in animal survival were observed. Hematoxylin and Eosin (H&E) staining showed that the tumors were virtually undetectable, and TUNEL staining revealed significant increases in apoptosis mediated by NDX.