Cytochrome C encapsulating theranostic nanoparticles: a novel bifunctional system for targeted delivery of therapeutic membrane-impermeable proteins to tumors and imaging of cancer therapy

Abstract

The effective administration of therapeutic proteins has received increased attention for the treatment of various diseases. Encapsulation of these proteins in various matrices, as a method of protein structure and function preservation, is a widely used approach that results in maintenance of the protein's function. However, targeted delivery and tracking of encapsulated therapeutic proteins to the affected cells is still a challenge. In an effort to advance the targeted delivery of a functional apoptosis-initiating protein (cytochrome c) to cancer cells, we formulated theranostic polymeric nanoparticles for the simultaneous encapsulation of cytochrome c and a near-infrared dye to folate-expressing cancer cells. The polymeric nanoparticles were prepared using a novel water-soluble hyperbranched polyhydroxyl polymer that allows for dual encapsulation of a hydrophilic protein and an amphiphilic fluorescent dye. Our protein therapeutic cargo is the endogenous protein cytochrome c, which upon cytoplasmic release, initiates an apoptotic response leading to programmed cell death. Results indicate that encapsulation of cytochrome c within the nanoparticle's cavities preserved the protein's enzymatic activity. The potential therapeutic property of these nanoparticles was demonstrated by the induction of apoptosis upon intracellular delivery. Furthermore, targeted delivery of cytochrome c to folate-receptor-positive cancer cells was achieved via conjugation of folic acid to the nanoparticle's surface, whereas the nanoparticle's theranostic properties were conferred via the coencapsulation of cytochrome c and a fluorescent dye. Considering that these theranostic nanoparticles can carry an endogenous cellular apoptotic initiator (cytochrome c) and a fluorescent tag (ICG) commonly used in the clinic, their use and potential translation into the clinic is anticipated, facilitating the monitoring of tumor regression.

Figure 1

Characterizations of the aliphatic HBPH polymer 4 and HBPH nanoparticles (HBPH NPs). A) Gel Permeation Chromatographic (GPC) trace of the polymer 4, showing the formation of high molecular weight polymer. B) Determination of the hydrodynamic diameter of the as-synthesized HBPH nanoparticles (Cyt C-HBPH NPs) using Dynamic Light Scattering (DLS: D = 63±2 nm, PDI = 0.73). C) Comparison between the UV/Vis absorption spectrum of Cyt C encapsulating nanoparticles (Cyt C-HBPH NPs) with that of free Cyt C and HBPH nanoparticles (HBPH NPs) itself, in solution. D) UV/Vis absorbance spectra of folate functionalized DiI and Cyt C encapsulating HBPH nanoparticles.

Figure 2

A) Steady state kinetic assay and the double reciprocal plot of oxidase activity of Cytochrome c encapsulating HBPH nanoparticles. B) TMB assay: Retention of Cyt C-HBPH nanoparticles peroxidase activity upon encapsulation, indicating that the encapsulated Cyt C protein remained active inside the polymer matrix. Each well contains an increasing amount (from left to right, 8, 9,10,11, 12, 13, 14, 15 µL) of Cyt C HBPH nanoparticle (0.71 µg of Cyt C/mL of HBPH nanoparticle), which translate into an increasing rate of oxidation of the colorimetric TMB substrate (from blue to yellow)

Figure 3

Release profiles of Cyt C from the protein encapsulating HBPH nanoparticles (Cyt C-HBPH NPs) at 37 °C. A) A slower rate of Cyt C release was observed in the presence of an esterase enzyme, while relatively B) faster release was observed at pH 4.0. No significant release of the encapsulated Cyt C was observed in PBS (pH 7.4). C) Catalytic activity of the released Cyt C [from esterase (i) and at pH 4.0 (iii)] visualized by the oxidation of TMB with hydrogen peroxide. Absence of TMB oxidation was observed in the PBS buffer (ii, iv; pH = 7.4).

Figure 4

In vitro retention of Cyt C-HBPH NP’s cell associated peroxidase activity. A) MCF 7 cells treated (6 h) with Cyt C-HBPH nanoparticle displayed peroxidase activity when the cell pellet was dissolved in TMB solution, indicated that the protein was still active upon release within the cell. B) images showing the blue color of the oxidized TMB solution compared to untreated.

Figure 5

Cell viability of MCF 7 cells treated with Cyt C encapsulating HBPH nanoparticles (Cyt C-HBPH NPs) compared to that of cells treated with the non-encapsulated nanoparticle (HBPH NPs) and non-treated cells (control). The protein encapsulating HBPH nanoparticles induced a significant reduction in the cell (MCF 7) viability after 6 h of incubation, where the non-encapsulated nanoparticle (HBPH NPs) showed no significant cytotoxicity. Control cells (MCF 7) were treated with 1× PBS. Average values of four measurements were depicted ± standard error.

Figure 6

MCF 7 cells showing the cytotoxic effect of the Cyt C encapsulating HBPH nanoparticles. Cells incubated with non-encapsulated nanoparticles (HBPH NPs) showed no cell death (C and D) similar to control cells (A and B). Cyt C encapsulating HBPH nanoparticles (Cyt C-HBPH NPs) induced significant cellular death as very less number of cells was found adhering to the plate (E) and an increase number of cell were found floated (F).

Figure 7

Detection of apoptotic cells. Induction of apoptosis observed when MCF 7 cells were treated with Cyt C encapsulating HBPH nanoparticles (Cyt C-HBPH NPs) and stained with DAPI and propidium iodide (PI) after 6 h of incubation. The arrow showed nuclear fragmentation indicating apoptosis.

Figure 8

Assessment of folate-HBPH nanoparticle uptake by human lung carcinoma A549 cells (A–I) and human breast adenocarcinoma MCF 7 cells (J–L) via confocal laser-scanning microscopy. Enhanced cellular internalization was observed upon incubation of the folate conjugated DiI-encapsulating HBPH nanoparticles (Fol-DiI-HBPH NPs) with A549 cells (A–C). In contrast, A549 cells pre-incubated with free folic acid showed minimal uptake (D–F), confirming receptor-mediated internalization. Meanwhile, A549 cells incubated with the Cyt C and DiI co-encapsulating folate-functionalized HBPH nanoparticles (Fol-Cyt C/DiI-HBPH NPs) induced significant cell death (G–I). As expected, no internalization of the Fol-DiI-HBPH NPs was observed in the MCF 7 as these cells are folate-receptor negative (J–L).

Figure 9

Detection of apoptotic cells. A) Selective induction of apoptosis observed in A549 cells (folate-receptor positive) and B) no cellular apoptosis observed in MCF 7 cells (folate-receptor negative) when incubated with folate conjugated Cyt C encapsulating HBPH nanoparticles (Fol-Cyt C-HBPH NPs), stained with DAPI and propidium iodide (PI) after 6 h of incubation.

Figure 10

UV-Vis absorbance spectra of the folate conjugated HBPH nanoparticles encapsulated with ICG (Fol-ICG-HBPH NPs) and Cyt C (Fol-Cyt C/ICG-HBPH NPs), showing absorbance at 355 nm, 414 nm and at 835 nm for folic acid, encapsulated Cyt C and ICG dye, respectively. Inset: Fluorescence emission spectra of the Fol-ICG-HBPH nanoparticles showing emission maximum at 865 nm. Similar results obtained from the corresponding Cyt C encapsulating HBPH nanoparticles.

Figure 11

Near infrared fluorescence images of the ICG encapsulating HBPH nanoparticles (Fol-ICG-HBPH NPs) incubated with A) MCF 7 and B) A549 cells, indicating receptor-mediated internalizations of our theranostic HBPH nanoparticles. Images taken from a Xenogen IVIS system using ICG filter set. Each well contains an increasing amount (from left to right, 20, 40, 60 80 µL) of Fol-ICG-HBPH NPs (3.2 mg/mL), which translate into an increasing uptake of nanoparticles and higher fluorescence emission.

Scheme 1

Schematic representation of the synthesis of three dimensional hydrophilic HBPH polymer (4) and its functional HBPH nanoparticles (HBPH NPs). The monomer 3 with three-bond connectivity polymerized three-dimensionally, resulting in a highly branched polymer with amphiphilic cavities in the polymeric structure.