A self-assembly and stimuli-responsive fusion gelonin delivery system for tumor treatment
Introduction
Of the current distinct treatment options, chemotherapy remains to be a major component of all cancer therapeutic regimens. However, the efficacy of chemotherapy is limited due to its side-effects associated with drug dosing, and poor effect of drug accumulation in the tumor target site. Macromolecules have attracted a strong interest due to their greater efficiency and selectivity, but many problems such as poor bioavailability, high instability in physiological environment, unpredictable toxicity and low ability at endocytosis [1] have still persisted.
Ribosome-inactivating proteins (RIPs) have attracted much attention due to their high anti-tumor activity induced by ribosome inactivation-related cytotoxicity [2]. RIPs depurate the 28S rRNA of the 60S ribosome subunit by their N-glycosidase enzymatic activity, and block the metabolism by inhibiting protein synthesis, slowly triggering the irreversible cell death [3]. Due to high substrate specificity and high tumor cell toxicity, RIPs have been marked as potent protein toxins for cancer therapy, and some kinds of RIPs have already been developed as potential anti-tumor drugs and have undergone various clinical trials [4]. Gelonin is a typical example of RIPs, which is formed by a single chain glycoprotein with about 30 kDa molecular weight [5]. However, the therapeutic potential of gelonin has not been clinically confirmed due to its low endocytic capacity.
Cell-penetrating peptides (CPPs) have been proved to be an efficient tool for intracellular delivery, and are widely used to transport drugs, proteins, nucleic acids, liposomes, imaging agents, peptides, quantum dots or nanocarrier drugs [6], [7], [8]. Low-molecular-weight protamine (LMWP), a well-known CPP which derived from protamine [9], [10], and is associated with high efficacy in the delivery of cargoes, especially endocytosis of macromolecule payloads [11]. In most of the cases, the peptide is conjugated with the cargo by chemical method [12], [13], [14]. Recombinant strategies have been employed to develop proteins fused with CPP peptides in order to establish an effective and stable method for protein penetration [15].
The stimuli-responsive tumor-targeting drug delivery systems assist in enhancing the specificity and local drug concentration [16]. Enzyme-activatable drug delivery system, which is a subtype of stimuli-responsive system, has been designed to lower the off-target activation of the delivered CPPs [17]. Among several varieties of enzymes, matrix metalloproteinase-2 (MMP-2) has been deeply investigated in tumor diagnosis and disease control [18], and is shown to over-express in diversified cancer types [19]. Therefore, a combination of CPP-based drug delivery system with MMP-2 enzyme-responsive system could overcome the hurdle of macromolecular drug delivery [20].
The biomacromolecule drugs when administered intravenously cause blood opsonization and overcome tumor microenvironment barriers. These are generally degraded of varied enzymes in the plasma and rapidly cleared the reticuloendothelial system (RES). They are also less exposed in tumor sites [21]. In recent years, several researchers have paid attention to nanoparticles that assist in conjugating or encapsulating the biomacromolecule drugs for crossing the barriers. It is important that sufficiently high doses of nanoparticles should be administered for the diseased cells and tissues [22], [23]. Enhanced permeability and retention (EPR) effect has been investigated in the past decades, which is considered as a new approach to gain high drug accumulation in tumor-targeting sites. Researchers have found that the capillary walls are leaky and incomplete to allow nanoparticles to pass through and accumulate in the targeting tumor areas. Magnetic iron oxide nanoparticles (MIONs) are deeply investigated their potential as imaging probes or drug carriers because of their superparamagnetic behavior [24]. The use of EPR effects as passive approaches for accumulation of nanoparticles in the tumor region through leaky vasculature have exploited with immunogenic cell death (ICD) inducers previously [25]. MION based drug delivery systems could also be used for magnetic guiding. Firstly, the surface of MIONs should be coated with materials that provide functional groups for further modification. Secondly, the formulation of delivery systems facilitates drug loading as well as biocompatibility simultaneously. Nickel (Ni)-modified MIONs (Fe2+ replaced by Ni2+) fit these points as drug carriers for magnetic guided drug delivery applications and have become a potential solution for in vivo targeting [26]. Over the past decades, a short chain polypeptide which contains six His-residues (His tag) were generally used for obtaining pure recombinant proteins. Among these potential drug carriers, Nickel (Ni)-modified MIONs is one of the best choices for the chelate bonds between the Ni2+ and imidazole from His tag.
We herein developed an enzyme-activated system to enhance the specificity of drug delivery with CPP to overcome the barrier of endocytosis. Also, a magnetic guided protein toxin delivery system has been designed to improve the therapeutic efficacy, along with EPR effects to lower the improper accumulation of drug in normal tissues and to increase the concentration in tumor regions due to leaky capillary walls. Initially, an optimized engineered fusion strategy for protein toxin drug development has been introduced. The recombinant peptide is then fused at gelonin C-terminus with gene-editing technology, including a LMWP, an MMP-2 substrate peptide (MSP) and His tag for marking and synthesis. The nickel column is used for the isolation and elution of the His-tagged biomacromolecules. The advantage of conjugation and purification by using this technique is faster in time and easier in operation. Secondly, the fused proteins (rGel) were self-assembled to Ni-MIONs (i.e. nickel ferrite) via automatic chelation (electron interaction activity) between the six His-residues amino acids and nickel ions, becoming a completed form of drug delivery system: rGel-NiFe2O4. In this case, the rGel carried by Ni-MIONs showed no cell-penetration activity because of the masking effect of carriers and unexposed enzyme cutting site till the enzyme-activating system works in the tumor microenvironment. Thirdly, after activation by tumor associated over-expressed MMP-2, the MSP linker was cut down, the LMWP-Gelonin was released, and reinstalled the pharmacological activity. This is a potential modular solution for delivering biomacromolecules, demanding the biocompatibility and cell membrane penetration. Drug accumulation in the tumor region and inhibition of tumorigenesis are anticipated using the drug delivery system rGel-NiFe2O4 (Scheme 1).
Section snippets
Materials
Tryptone, yeast extract, agar and BCA protein quantitation kit were purchased from Aladdin (Shanghai, China). NaCl, NiCl2·6H2O, and FeCl3·6H2O were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Kanamycin, Isopropyl-β-d-1-thiogalactopyranoside (IPTG), Trimethyl-aminomethane hydrochloride (Tris-HCl) and imidazole were purchased from Sigma–Aldrich (St Louis, MO, USA). Escherichia coli (E. coli) BL21 (DE3) competent cells were purchased from ComWin Biotech (Beijing, China).
Purification and characterization of fusion proteins
The expressed protein chimera rGel contained the RIP gelonin, a linker with the LMWP peptide, MMP-2 specific cleavage site, and His tag. To preserve the LMWP penetration and gelonin activities in rGel, the LMWP domain was fused with the N-terminus of the gelonin domain through a flexible GGGGS (Gly× 4-Ser) linker. As a control protein rGel-M (m for mutated), the MMP-2 cleavage site PLGVR (Pro-Leu-Gly-Val-Arg) on rGel was mutated to PLGKL (Pro-Leu-Gly-Lys-Leu), leading to the sequence
Conclusion
In summary, a potential prodrug mechanism as delivery system has been developed to overcome difficulties in the delivery of antineoplastic macromolecular fusion proteins, which involves short half-life, severe immunogenicity and inefficient tissue distribution. The fusion protein rGel contained three additional functional domains including LMWP, MMP-2 specific substrate and His-tag for self-assembly. The His-tagged protein renders specific conjugation of nickel (Ni)-modified MIONs via chelation
Acknowledgements
This work was supported by Natural Science Foundation of Tianjin (NO18JCQNJC80700) and National Natural Science Foundation of China (81503019).
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