Advance of DNA and CCPs-based nanocarriers in drug delivery systems

2.1. Cell entry of DNA nanostructure and escape from enzymatic degradation

As we know, cell surface is composed by negatively charged glycoproteins and glycolipids, so that DNA is difficult to penetrate the cell membrane with the negative surface charge [9]. On the other hand, because of the large molecular weight, DNA cannot easily pass through biological membranes. Furthermore, the large size of the DNA greatly limits the diffusion in the extracellular space and extravasation in tissues with a continuous endothelium [10–12]. Due to its high chain flexibility, single-stranded DNA (ssDNA) is capable of self-matching and folding into unique tertiary structures [13], which make DNA nanostructure can readily pass through the cell membrane, though the cell entry mechanism for DNA origami nanostructures has not been clarified completely [14]. Liang and co-workers using a single-particle tracking method found that tetrahedral DNA nanostructure were internalized by mammalian cells via a caveolin-dependent pathway, according to the biochemical and fluorescence imaging experiments, they also demonstrated that DNA tetrahedra functionalized with a nuclear localization signal (NLS) could make them localized precisely in the cells, and escape from lysosome and nuclease, which have a potency as nonviral drug delivery carriers for targeted therapy [15]. To facilitate delivery of DNA origamis into the human cells, virus capsid proteins were employed to contact with DNA origamis through electrostatic interactions, it was indicated that the cellular transfection efficiency was significantly improved with the proteins concentrations increasing [16].

With the purpose of preventing the self-assembled DNA nanostructures from being degraded and facilitating their membrane permeability as drug delivery vehicles, DNA has been wrapped into polymeric particles, such as liposomes and nanospheres. With the protection of these particles, DNA was able to avoid being degraded by nuclease, which endow DNA with increased stability. DNA nanostructure is a negatively charged material and hard to cross the plasma membrane, therefore cationic compounds were applied to form a complex with DNA by an electrostatic interaction. So the complexes were eventually positively charged, which can combine with negatively charged cell membrane and finally be internalized.

2.2. DNA as nanocarriers for drug delivery

Because of the invention of DNA origami and the steerable properties, researchers are very interested in its potential application as intelligent drug carriers [17]. DNA origami nanostructures have become the promising targeted drug delivery nanocarriers as its unique advantages, including the biocompatibility, highly-effective self-assembly, highly-controllable nanostructure, addressable modification sites, and easy to load with biomolecules, such as anticancer drugs, imaging dyes, and nucleic acids and so on [18]. Recently, a large number of articles about the DNA nanostructures as molecular delivery carriers in diagnostics and therapeutics have been reported and proposed. For instance, some anticancer drugs have no effect on cancer cells, this phenomenon can be defined as multidrug resistance (MDR). MDR is a major obstacle to the successful treatment of cancer. Kim and co-workers demonstrated that the doxorubicin loaded in DNA nanostructures is considerably cytotoxic, while the unloaded DOX is virtually non-cytotoxic for the drug-resistant cells [19].

The most challenging application of these DNA nanostructures is to design a highly tunable and controllable delivery system, which is an efficient strategy in developing smart carriers with the ability to release the therapeutics in target lesion areas. It is a highly versatile biological polymer that can be manipulated precisely down to the molecular level using techniques from the existing molecular biology toolbox. For example, Gu et al. have developed a new ATP-mediated controlled drug release system comprised of a 2D nanomaterial (graphene oxide, GO) assembled nanoaggregates crosslinked by ATP-responsive DNA strands, when the intracellular ATP concentration is different from the extracellular environment, the controlled release of drugs is achieved [20,21].

A kind of nanospheres (NS) is composed of multifunctional and programmable DNA nanostructures, with monodispersity, dense compaction and uniform size. NSs exhibited high payload capacity and excellent biocompatibility [22]. Roh et al. employed X-shaped DNA (X-DNA) to prepare DNA nanospheres via photopolymerization, the size of the nanospheres was precisely controlled by regulating the concentration of the X-DNA monomers. The DNA building blocks photoassembled into nanospheres with well monodisperse, an extremely high crosslinking efficiency and yield. In addition, the anticancer drug doxorubicin was encapsulated in DNA nanospheres, and the results exhibited a high drug loading efficacy of 62%, however, the free drug showed no cytotoxicity to mammalian cells. This novel DNA nanomaterials with the advantages of highly tunability and controllability that make them adaptive in drug delivery applications [23].

Due to the special nanostructures, DNA possess the highly specific molecular recognition properties, which could be used as targeted drug delivery carriers and novel biosensors [24]. Luo et al. have utilized Y-DNA as the modular donor and X-DNA was acceptor molecule, then formed a molecular building block called ABC monomers by connecting the X-DNA to the Y-DNA. The ABC monomers, with multifunctional moieties, was a modular, branched, and anisotropic nanomaterials. They have demonstrated that ABC monomer could be used for highly-specific and ultrasensitive detection of pathogens. When the targeted pathogen DNA was present, a photo-crosslinked detectable polymeric spheres occurred. These target-driven polymer can serve as multi-drug delivery vectors due to their diverse functional groups, the internalization mechanism of the polymeric spheres was detected as actin-dependent endocytosis [25].

3.1. Mechanisms of CPPs uptake

Bioactive molecules can be grafted to nanoparticles, but a deficiency of them is unable to cross the lipid membranes of cells, it can greatly limit their application both in vitro and in vivo. Therefore, nanoparticles are often used to connect with CPPs to circumvent this cell-penetrating obstacle. The major advantage of CPPs is their ability to transport cargo to intracellular compartments of the cell. Nevertheless, the mechanisms of CPPs into cells is still a controversial issue, and not entirely elucidated. It is generally accepted that most CPPs and CPP-cargo complexes enter cells through endcytosis which can be divided into four categories: macropinocytosis, clathrin-mediated endcytosis, caveolae mediated endocytosis, and clathrin/caveolae-independent endocytosis [31]. With the purpose of observing the pathways of endocytic uptake, the methods mainly rely on the flow cytometry and live cell scanning confocal microscopy, the former is used to detect the number of CPPs taken up into cells through the intensity of fluorescence, and the latter is able to monitor the specific intracellular localization of CPPs or the connected cargo molecules internalized by cells [32]. Another cell entry mechanism proposed is energy-independent direct translocation. A hydrophilic, charged peptide could directly migrate through the hydrophobic lipid bilayer. It suggested that direct penetration was the uptake mechanism for most CPPs, such as TAT, Antennapedia, poly-Arg, Transportan, MPG and Pep-1 and so forth [33].

3.2. Smart CPPs-mediated nanocarriers for drug delivery

CPPs can now provide an effective means of intracellular drug delivery, however, they also display some shortcomings. On the one hand, the stability in vivo of these peptides, which is on the hazard in the circulation and in the cell cytoplasm. These peptides can be enzymatically cleaved by proteolytic enzymes before they reach their target regions. Therefore, it’s necessary to obtain multifunctional drug delivery systems with steric protection of CPP moieties or synthesis of protease-resistant peptides against proteolytic degradation. The integrity of CPPs is essential to ensure the function of efficient penetration. V.P. Torchilin and co-workers explored the proteolytic cleavage of TATp on the surface of TATp-modified liposomes and micelles, they discovered that these TATp-micelles were modified by the addition of longer PEG-PE blocks, protecting TATp from attacking by the steric hindrances of these blocks [34,35].

On the other hand, CPPs have no cell type specificity and can enter any cell they come in contact with. This lack of selectivity put the normal tissues at risk because of drug-induced toxic effect. Compared with normal tissue, the pH value of pathologic region is lower, which provide a unique cellular environment to the target drug delivery. Kale et al. have prepared a PEGylated liposomes through the pH-sensitive hydrazone between them, and the liposomes are modified with TAT peptide (TATp), pH-sensitive PEG blocks act as a barrier to protect the peptide. Because of the low pH in tumor sites, the PEG coating is lost fast and exposed TAT peptides, which provide an efficient penetration into the tumor cells [36,37]. Wang and co-workers synthesized the bioactive polymer poly(L-glutamic acid)_n-b-poly(D, L-lactic acid)_m and use it to form doxorubicin-loaded hybrid polymeric micelles with pH-responsive conformation transition. These pH-responsive characteristics were used to control the functionalities of the micelles and to target localization. The poly(L-glutamic acid)_n-b-poly(D, L-lactic acid)_m protected Tat peptides from proteolysis during circulation. Under tumor-acidic conditions, the structural changes of the polymeric micelles with shorter poly(L-glutamic acid) blocks formed channels to promote the release of doxorubicin [38]. Temperature responsiveness has been identified as the key to achieve the site-specific targeting of drugs. Thermo-responsive microgel capsules (MCs) are promising candidate for drug delivery due to their biocompatibility, biodegradability, and non-immunogenicity and stimuli-responsive characteristics. Temperature responsiveness has been certified as a significant method to accumulated Elastic-like polypeptides (ELPs) in tumors and the site-specific targeting can facilitate drug delivery [39]. Cheng and co-workers successfully prepared a series of thermo-responsive ELP/Bovine serum albumin (BSA) microgel capsules (MCs), and observed the diameters changes of the MCs with different ratio over a temperature range between 20 and 40°C. Thermally induced changes in volume decreased when the ratio of ELP to BSA decreased. The MCs without ELP kept the volume unchanged and no pore formation. When the environmental temperatures were higher than the lower critical solution temperature (Tt), the pore windows both at the surface and inside the shell were opened, accelerating the loading efficiency and release rate. The pore windows were closed with the temperatures decreased below Tt for 20 h, slowing the release rate of rhodamine B [40].