The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.
We have developed doxorubicin (DOX)-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (DP) conjugated with polyethylene glycol (PEG) and transferrin (Tf) to form Tf-PEG-DPs (TPDPs), and incorporated these TPDPs into three-dimensional (3-D) PLGA porous scaffolds to form a controlled delivery system. To our knowledge, this represents the first use of a Tf variant (oxalate Tf) to improve the targeted delivery of drug-encapsulated nanoparticles (NPs) in PLGA scaffolds to PC3 prostate cancer cells. The PLGA scaffolds with TPDPs incorporated have been shown to release drugs for sustained delivery and provided a continuous release of DOX. The MTS assay was also performed to determine the potency of native and oxalate TPDPs, and a 3.0-fold decrease in IC50 values were observed between the native and oxalate TPDPs. The lower IC50 value for the oxalate version signifies greater potency compared to the native version, since a lower concentration of drug was required to achieve the same therapeutic effect. These results suggest that this technology has potential to become a new implantable polymeric device to improve the controlled and targeted drug delivery of Tf-conjugated NPs for cancer therapy.
Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer–specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.
Targeted killing of cancer cells by engineered nanoparticles holds great promise for noninvasive photothermal therapy applications. We present the design and generation of a novel class of gold nanoshells with cores composed of self-assembled block copolypeptide vesicles with photothermal properties. Specifically, poly(L-lysine)60-block-poly(L-leucine)20 (K60L20) block copolypeptide vesicles coated with a thin layer of gold demonstrate enhanced absorption of light due to surface plasmon resonance (SPR) in the near-infrared range. We show that the polypeptide-based K60L20 gold nanoshells have low toxicity in the absence of laser exposure, significant heat generation upon exposure to near-infrared light, and, as a result, localized cytotoxicity within the region of laser irradiation in vitro. To gain a better understanding of our gold nanoshells in the context of photothermal therapy, we developed a comprehensive mathematical model for heat transfer and experimentally validated this model by predicting the temperature as a function of time and position in our experimental setup. This model can be used to predict which parameters of our gold nanoshells can be manipulated to improve heat generation for tumor destruction. To our knowledge, our results represent the first ever use of block copolypeptide vesicles as the core material of gold nanoshells.
We previously investigated the intracellular trafficking properties of our novel poly(l-glutamate)60-b-poly(l-leucine)20 (E60L20) vesicles (EL vesicles) conjugated to transferrin (Tf). In this study, we expand upon our previous work by investigating the drug encapsulation, release, and efficacy properties of our novel EL vesicles for the first time. After polyethylene glycol (PEG) was conjugated to the vesicles for steric stability, doxorubicin (DOX) was successfully encapsulated in the vesicles using a modified pH-ammonium sulfate gradient method. Tf was subsequently conjugated to the vesicles to provide active targeting to cancer cells and a mode of internalization into the cells. These Tf-conjugated, DOX-loaded, PEGylated EL (Tf-DPEL) vesicles exhibited colloidal stability and were within the allowable size range for passive and active targeting. A mathematical model was then derived to predict drug release from the Tf-DPEL vesicles by considering diffusive and convective mass transfer of DOX. Our mathematical model reasonably predicted our experimentally measured release profile with no fitted parameters, suggesting that the model could be used in the future to manipulate drug carrier properties to alter drug release profiles. Finally, an in vitro cytotoxicity assay was used to demonstrate that the Tf-DPEL vesicles exhibited enhanced drug carrier efficacy in comparison to its non-targeted counterpart.