Targeted Delivery of Nanocarrier-Conjugated Doxorubicin to Widen the Therapeutic Window of the Most Active Drug in Lymphoma Therapeutics
Authors: Arumov Artavazd , Liyanage Piumi Y. , Trabolsi Asaad , Li Lingxiao , Roberts Evan R. , Bilbao Daniel , LeBlanc Roger M. , Schatz Jonathan H. 
Published: November 2019

Doxorubicin (Dox) remains the most active drug against aggressive lymphomas, forming the backbone of multiple potentially curative frontline combination regimens. R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), for example, cures diffuse large B-cell Lymphoma (DLBCL), the most common lymphoid malignancy in the United States, greater than 60% of the time. Patients with relapsed/refractory disease, however, have poor prognosis and require new options. Advances in nanotechnology provide new opportunities to widen therapeutic windows for existing drugs by enhancing delivery to tumor cells and limiting toxicities to non-malignant tissues. Carbon-Nitride Dots (CND) are novel nanocarriers we have developed that can be conjugated with a diverse range of molecules and have an established safe pharmacologic profile. Here, we sought CND-based enhancement of Dox's anti-lymphoma activities.
We generated unconjugated CNDs (~3 nm) through a hydrothermal microwave synthesis, followed by carbodiimide cross-linking bioconjugation steps to covalently link Dox and/or transferrin (TF), the protein ligand for the transferrin receptor (TFR). Because TFR is expressed on the cell surface in a range of B-cell lymphomas including DLBCL, we aimed to increase Dox delivery to tumor cells and limit delivery to non-malignant tissues. We probed a cohort of DLBCL cell lines for TFR expression via western blot, followed by baseline viability assays. Unconjugated CNDs showed no toxicity in vitro, while conjugation to Dox alone resulted in potency similar to unconjugated Dox. CND-Dox-TF, however, was 1-2 Log10 more potent than Dox alone (LD50 1.2-48.5 nM vs. 205.5->1000 nM), consistent with enhanced activity due to the entry of the nanocarrier into the cells through the TF-TFR interaction. To create a functional model, we cloned the TFR1 gene into a GFP-lentiviral expression vector. We infected previously tested cell lines with TFR1, confirmed increased TFR expression via western blot, and exposed cells to CND nanocarriers. TFR1-infected cells showed selective disadvantage during CND-Dox-TF treatment compared to uninfected and empty-vector controls, while CND and CND-Dox controls showed no differential effect. After establishing CND-Dox-TF proof of principle in vitro, we next initiated testing in vivo, beginning with maximum-tolerated dose (MTD) finding experiments. We treated three groups of non-tumor-bearing NOD scid gamma (NSG) mice intravenously with a single dose of (1) CND, (2) CND-Dox, and (3) CND-Dox-TF. Maximum dosing of CND-Dox-TF based on solubility in 100 µL PBS was roughly 1:4 molar equivalent to Dox MTD (3.3 mg/kg). This dose of CND-Dox-TF caused decreased body weight to >20% loss from starting dosing and animals had to be sacrificed, with organ pathology pending. Molar equivalent dosing of CND and CND-Dox, however, resulted in no weight loss, demonstrating biologic activity of the TF moiety but unfortunately intolerable toxicity at this initial dose. We then reduced the CND-Dox-TF dose to 1:16 molar equivalent to Dox MTD and repeated dosing to three non-tumor-bearing animals. This resulted in decreased body weight of 10% from starting dose by day 11, followed by a rebound to normal body weight by day 17. This observed body weight fluctuation is similar to what is seen under a 0.75% of Dox MTD.
With a dose of 33 mg/kg identified as the MTD of the CND-Dox-TF, we are now proceeding to anti-tumor efficacy experiments in four available PDX models of DLBCL (two each from previously untreated and relapsed/refractory patients). We will compare the MTD of Dox versus the MTD of CND-Dox-TF initially as single agents. We will then compare R-CHOP to "R-nanoCHOP" (replacement of Dox with the MTD of CND-Dox-TF). We hypothesize that both through the TF-TFR interaction and enhanced tumor permeability and retention that are known properties of CNDs, CND-Dox-TF treated mice will have improved anti-lymphoma responses. In sum, we show that a TF ligand conjugated to Dox via our CND nanocarrier significantly increases the anti-lymphoma efficacy of Dox on DLBCL cell lines. Importantly, we also show these novel nanocarrier therapeutic molecules are safe to administer in vivo, and we define MTD for studies moving forward. Proof of principle that CND nanotechnology enhances anti-lymphoma activity of Dox would open the door for many such approaches aimed at a variety of malignancies.

Disclosures
No relevant conflicts of interest to declare.