Encapsulation of copper phenanthroline within horse spleen apoferritin: characterisation, cytotoxic activity and ability to retain temozolomide.

Protein capsules are promising drug delivery vehicles for cancer research therapies. Apoferritin (AFt) is a self-assembling 12 nm diameter hollow nanocage with many desirable features for drug delivery, however, control of drug retention inside the protein cage remains challenging. Here we report the encapsulation of copper(ii)-1,10-phenanthroline (Cu(phen)) within the horse spleen AFt (HSAFt) nanocage, by diffusion of the metal through the pores between the protein subunits. Transmission electron microscopy revealed the formation of organised copper adducts inside HSAFt, without affecting protein integrity. These structures proved stable during storage (>4 months at -20 °C). Exposure to physiologically relevant conditions (37 °C) showed some selectivity in cargo release after 24 h at pH 5.5, relevant to the internalisation of AFt within the endosome (60% release), compared to pH 7.4, relevant to the bloodstream (40% release). Co-encapsulation of temozolomide, a prodrug used to treat glioblastoma multiforme, and Cu(phen) enabled entrapment of an average of 339 TMZ molecules per cage. In vitro results from MTT and clonogenic assays identified cytotoxic activity of the Cu(phen), HSAFt-Cu(phen) and HSAFt-Cu(phen)-TMZ adducts against colorectal cancer cells (HCT-116) and glioblastoma cells (U373V, U373M). However, the presence of the metal also contributed to more potent activity toward healthy MRC5 fibroblasts, a result that requires further investigation to assess the clinical viability of this system.

Correction to "Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy".

[This corrects the article DOI: 10.1021/acsomega.2c00997.].

Wireless electrical-molecular quantum signalling for cancer cell apoptosis.

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments.

Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy.

The O-acetyl (or acetate) derivative of the Aspidosperma alkaloid Jerantinine A (JAa) elicits anti-tumor activity against cancer cell lines including mammary carcinoma cell lines irrespective of receptor status (0.14 < GI50 < 0.38 µM), targeting microtubule dynamics. By exploiting breast cancer cells' upregulated transferrin receptor 1 (TfR1) expression and apoferritin (AFt) recognition, we sought to develop an AFt JAa-delivery vehicle to enhance tumor-targeting and reduce systemic toxicity. Optimizing pH-mediated reassembly, ∼120 JAa molecules were entrapped within AFt. Western blot and flow cytometry demonstrate TfR1 expression in cancer cells. Enhanced internalization of 5-carboxyfluorescein-conjugated human AFt in SKBR3 and MDA-MB-231 cancer cells is observed compared to MRC5 fibroblasts. Accordingly, AFt-JAa delivers significantly greater intracellular JAa levels to SKBR3 and MDA-MB-231 cells than naked JAa (0.2 µM) treatment alone. Compared to naked JAa (0.2 µM), AFt-JAa achieves enhanced growth inhibition (2.5-14-fold; <0.02 µM < GI50 < 0.15 µM) in breast cancer cells; AFt-JAa treatment results in significantly reduced clonal survival, more profound cell cycle perturbation including G2/M arrest, greater reduction in cell numbers, and increased apoptosis compared to the naked agent (p < 0.01). Decreased PLK1 and Mcl-1 expression, together with the appearance of cleaved poly (ADP-ribose)-polymerase, corroborate the augmented potency of AFt-JAa. Hence, we demonstrate that AFt represents a biocompatible vehicle for targeted delivery of JAa, offering potential to minimize toxicity and enhance JAa activity in TfR1-expressing tumors.

Apoferritin and Dps as drug delivery vehicles: Some selected examples in oncology.

BACKGROUND: The ideal nanoparticle should be able to encapsulate either pharmaceutical agents or imaging probes so that it could treat or image clinical tumours by targeting the cancer site efficiently. Further, it would be an added advantage if it demonstrates: small size, built in targeting, biocompatibility and biodegradability. Ferritin, which is an endogenous self-assembling protein, stores iron and plays a role in iron homeostasis. When iron atoms are removed apoferritin (AFt) is formed which consists of a hollow shell where it can be used to load guest molecules. Due to its unique architecture, AFt has been investigated as a versatile carrier for tumour theranostic applications. DNA-binding protein from starved cells (Dps), which also belongs to the ferritin family, is a protein found only in prokaryotes. It is used to store iron and protect chromosomes from oxidative damage; because of its architecture, Dps could also be used as a delivery vehicle. CONCLUSIONS: Both these nano particles are promising in the field of oncology, especially due to their stability, solubility and biocompatibility features. Further their exterior surface can be modified for better tumour-targeting ability. More studies, are warranted to determine the immunogenicity, biodistribution, and clearance from the body. GENERAL PERSPECTIVE: This review discusses a few selected examples of the remarkable in vitro and in vivo studies that have been carried out in the recent past with the use of AFt and Dps in targeting and delivery of various pharmaceutical agents, natural products and imaging probes in the field of oncology.

Chemosensitization of Temozolomide-Resistant Pediatric Diffuse Midline Glioma Using Potent Nanoencapsulated Forms of a N(3)-Propargyl Analogue.

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O6-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether ß-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC50 = 14.6 (Lip-N3P) vs 32.8 µM (N3P); DIPG-IV) and (IC50 = 101.8 (AFt-N3P) vs 111.9 µM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT+ DIPG-VI (100 µM) and mismatch repair deficient DIPG-XIX (50 µM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (89Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

New Treatments in Renal Cancer: The AhR Ligands.

Kidney cancer rapidly acquires resistance to antiangiogenic agents, such as sunitinib, developing an aggressive migratory phenotype (facilitated by c-Metsignal transduction). The Aryl hydrocarbon receptor (AhR) has recently been postulated as a molecular target for cancer treatment. Currently, there are two antitumor agent AhR ligands, with activity against renal cancer, that have been tested clinically: aminoflavone (AFP 464, NSC710464) and the benzothiazole (5F 203) prodrug Phortress. Our studies investigated the action of AFP 464, the aminoflavone pro-drug currently used in clinical trials, and 5F 203 on renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis and cell migration. Both compounds caused cell cycle arrest and apoptosis but only 5F 203 potently inhibited the migration of TK-10, Caki-1 and SN12C cells as well as the migration signal transduction cascade, involving c-Met signaling, in TK-10 cells. Current investigations are focused on the development of nano-delivery vehicles, apoferritin-encapsulated benzothiazoles 5F 203 and GW610, for the treatment of renal cancer. These compounds have shown improved antitumor effects against TK-10 cells in vitro at lower concentrations compared with a naked agent.

Delivery of Temozolomide and N3-Propargyl Analog to Brain Tumors Using an Apoferritin Nanocage.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 µM for AFt-TMZ, compared to 35 and 376 µM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.

Protein Encapsulation of Experimental Anticancer Agents 5F 203 and Phortress: Towards Precision Drug Delivery.

INTRODUCTION: Advancement of novel anticancer drugs into clinical use is frequently halted by their lack of solubility, reduced stability under physiological conditions, and non-specific uptake by normal tissues, causing systemic toxicity. Their progress to use in the clinic could be accelerated by the development of new formulations employing suitable and complementary drug delivery vehicles. METHODS: A robust method for apoferritin (AFt)-encapsulation of antitumour benzothiazoles has been developed for enhanced activity against and drug delivery to benzothiazole-sensitive cancers. RESULTS: More than 70 molecules of benzothiazole 5F 203 were encapsulated per AFt cage. Post-encapsulation, the size and integrity of the protein cages were retained as evidenced by dynamic light scattering. ToF-SIMS depth profiling using an argon cluster beam confirmed 5F 203 exclusively within the AFt cavity. Improved encapsulation of benzothiazole lysyl-amide prodrugs was achieved (~130 molecules of Phortress per AFt cage). Transferrin receptor 1, TfR1, was detected in lysates prepared from most cancer cell lines studied, contributing to enhanced anticancer potency of the AFt-encapsulated benzothiazoles (5F 203, Phortress, GW 610, GW 608-Lys). Nanomolar activity was demonstrated by AFt-formulations in breast, ovarian, renal and gastric carcinoma cell lines, whereas GI50 >50 µM was observed in non-tumourigenic MRC-5 fibroblasts. Intracellular 5F 203, a potent aryl hydrocarbon receptor (AhR) ligand, and inducible expression of cytochrome P450 (CYP) 1A1 were detected following exposure of sensitive cells to AFt-5F 203, confirming that the activity of benzothiazoles was not compromised following encapsulation. CONCLUSION: Our results show enhanced potency and selectivity of AFt-encapsulated 5F 203 against carcinomas derived from breast, ovarian, renal, colorectal as well as gastric cancer models, and offer realistic prospects for potential refinement of tumour-targeting and treatment, and merit further in vivo investigations.

Synthesis of folic acid functionalized gold nanoclusters for targeting folate receptor-positive cells.

We report on the synthesis of water-soluble gold nanoclusters capped with polyethylene glycol (PEG)-based ligands and further functionalized with folic acid for specific cellular uptake. The dihydrolipoic acid-PEG-based ligands terminated with -OMe, -NH2 and -COOH functional groups are produced and used for surface passivation of Au nanoclusters (NCs) with diameters <2 nm. The produced sub 2 nm Au NCs possess long-shelf life and are stable in physiologically relevant environments (temperature and pH), are paramagnetic and biocompatible. The paramagnetism of Au NCs in solution is also reported. The functional groups on the capping ligands are used for direct conjugation of targeting molecules onto Au NCs without the need for post synthesis modification. Folic acid (FA) is attached via an amide group and effectively target cells expressing the folate receptor. The combination of targeting ability, biocompatibility and paramagnetism in FA-functionalized Au NCs is of relevance for their exploitation in nanomedicine for targeted imaging.

Listeria innocua Dps as a nanoplatform for bioluminescence based photodynamic therapy utilizing Gaussia princeps luciferase and zinc protoporphyrin IX.

Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490 nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent.

Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells.

Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated ∼3-fold enhanced in vitro activity (GI50 = 79 µM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI50 > 200 µM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 µM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation.

In search of effective therapies to overcome resistance to Temozolomide in brain tumours.

Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease.

Development of novel apoferritin formulations for antitumour benzothiazoles.

BACKGROUND: The benzothiazole structure is important in medicinal chemistry, and 5-fluoro-2-(3,4-dimethoxyphenyl) benzothiazole (GW 610) is of particular interest as it shows outstanding anticancer activity in sensitive breast and colorectal carcinoma cell lines via generation of lethal DNA adducts in sensitive cancer cells. Despite promising activity, poor water solubility limits its applications. The apoferritin (AFt) protein cage has been proposed as a robust and biocompatible drug delivery vehicle. AIMS: Here, we aim to enhance solubility of GW 610 by developing amino acid prodrug conjugates and utilizing the AFt capsule as drug delivery vessel. METHODS AND RESULTS: The potent experimental antitumour agent, GW 610, has been successfully encapsulated within AFt with more than 190 molecules per AFt cage. The AFt-GW 610 complex exhibits dose-dependent growth inhibition and is more potent than GW 610 alone in 5/7 cancer cell lines. To enhance both aqueous solubility and encapsulation efficiency, a series of amino acid esters of GW 608 prodrug were synthesized via N,N'-dicyclohexylcarbodiimide ester coupling to produce molecules with different polarity. A dramatic increase in encapsulation efficiency was achieved, with more than 380 molecules of GW 608-Lys molecules per AFt cage. Release studies show sustained release of the cargo over 12 hours at physiologically relevant pH. The AFt-encapsulated amino acid modified GW 608 complexes are sequestered more rapidly and exhibit more potent anticancer activity than unencapsulated agent. CONCLUSION: These results indicate that AFt-encapsulation of GW 610 prodrug provides a biocompatible delivery option for this potent, selective experimental antitumour agent and for amino acid-modified GW 608. Of particular interest is the encapsulation efficiency and in vitro antitumour activity of AFt-GW 608-Lys, which warrants further preclinical evaluation.

An Apoferritin-based Drug Delivery System for the Tyrosine Kinase Inhibitor Gefitinib.

Anticancer drug Gefitinib encapsulated within human heavy chain apoferritin by diffusion allows pH-controlled sustained release of cargo. The combination of increased cellular uptake, and potent and enhanced antitumor activity against the HER2 overexpressing SKBR3 cell line compared to Gefitinib alone, makes it a promising carrier for delivery of drugs to tumor sites.

Apoferritin-encapsulated PbS quantum dots significantly inhibit growth of colorectal carcinoma cells.

Colorectal carcinoma (CRC) is the 3rd most common cancer worldwide, thus development of novel therapeutic strategies is imperative. Herein potent, selective dose-dependent antitumor activity of horse spleen apoferritin encapsulated PbS quantum dots (AFt-PbS) against two human-derived colorectal carcinoma cell lines is reported (GI50∼ 70 µg mL-1). Following in vitro exposure to AFt-PbS, CRC cells fail to recover proliferative capacity, and undergo apoptosis triggered by the generation of reactive oxygen species (ROS). In stark contrast, the AFt-PbS nanocomposites do not affect the growth and cell cycle of non-tumor human microvessel endothelial HMEC-1 cells (GI50 > 500 µg mL-1). In vivo, AFt-PbS QDs are well tolerated by mice. Neither adverse health nor behavioral indicators were observed throughout the 15 day study. The photoluminescence of AFt-PbS combined with selective antitumor activity offer potential development of AFt-PbS for simultaneous non-invasive imaging and treatment of malignant tissue.

Fabrication of polypyrrole nano-arrays in lysozyme single crystals.

A template-directed method for the synthesis and organization of partially oxidized polypyrrole (PPy) nanoscale arrays within the solvent channels of glutaraldehyde-cross-linked lysozyme single crystals is presented. Macroscopic single crystals of the periodically arranged protein-polymer superstructure are electrically conductive, insoluble in water and organic solvents, and display increased levels of mechanical plasticity compared with native cross-linked lysozyme crystals.