New drug delivery systems have been widely studied in these last years ( Drug delivery systems: An updated review, 2012.).
The discovery of innovative techniques for the manipulation of materials at the nano scale lead the science to break limits that before were considered impossible to overcome. Many new medical applications have been demonstrated by using hi-tech materials and techniques.
By providing enhanced efficiency, the clinicians would be able to release the correct dose of drug not only in the right tissue, but also at the correct cell. All this would lead to maximum increase of drug concentration with a very low intensity of side effects, preserving the healthy tissue.
The usefullness of this new technologies is shown for example in the revolving possibilities for the actual anti-cancer chemotherapy, in which their application would reduce the toxicity, helping in the improvement of the quality life of the patients.
Advanced materials and processing for drug delivery:the past and the future, 2013.
The research is focused on developing the most suitable molecules. The goals are to use carriers completely compatible with the patient body, meaning that they should not inducing immune reaction or overloading patient’s organs already weak for the disease. The pattern of the molecule would be able to be controlled by injecting it in the blood stream and after that by letting it completely on his own. The carriers have to be able to recognize the correct target and enter in it and release the correct amount of drug. There are many researches trying to figure out this challenge, and here I present 3 of the most interesting and successful solutions discovered so far.
BREAST CANCER AND LISOSOMES
Liposome-based drug delivery in breast cancer treatment, 2013.
The first application I’m going to present is about the treatment of breast cancer. Long circulating macromolecular carriers such as liposomes can exploit the enhanced permeability and retention’s effect for preferential extravasation from tumor vessels.
Liposomal anthracyclines have achieved highly efficient drug encapsulation, resulting in significant anticancer activity with reduced cardiotoxicity, and include versions with greatly prolonged circulation such as liposomal daunorubicin and pegylated liposomal doxorubicin. Pegylated liposomal doxorubucin has shown substantial efficacy in breast cancer treatment both as monotherapy and in combination with other chemotherapeutics. Additional liposome constructs are being developed for the delivery of other drugs.
As discussed, currently approved liposomal drug delivery systems provide stable formulation, provide improved pharmacokinetics, and a degree of ‘passive’ or ‘physiological’ targeting to tumor tissue. However, these carriers do not directly target tumor cells. The design modifications that protect liposomes from undesirable interactions with plasma proteins and cell membranes, and which contrast them with reactive carriers such as cationic liposomes, also prevent interactions with tumor cells.
Instead, after extravasation into tumor tissue, liposomes remain within tumor stroma as a drug-loaded depot. Liposomes eventually become subject to enzymatic degradation and/or phagocytic attack, leading to release of drug for subsequent diffusion to tumor cells. The next generation of drug carriers under development features direct molecular targeting of cancer cells via antibody-mediated or other ligand-mediated interactions.
LIPOSOME BASED CO-DELIVERY SYSTEM
ATP-responsive DNA-graphene hybrid nanoaggregates for anticancer drug delivery, 2015.
Liposome-based co-delivery system consisting of a fusogenic liposome encapsulating ATP-responsive elements and a liposome containing ATP for ATP-triggered drug release mediated by the liposomal fusion. The fusogenic liposome contains a protein–DNA complex core, which is composed of protamine and an ATP-responsive DNA scaffold (Figure 1a). The DNA segment was composed of the duplex hybridized by the ATP aptamer and its complementary single-stranded DNA, which shows a specific and high affinity to ATP and has been often utilized for ATP detection. Doxorubicin(DOX), a model small-molecule anticancer drug that is particularly prone to be intercalated in the GC pair of DNA motif, is applied to form the DOX-loaded duplex (DOX-Duplex).
The interaction of the complex of the ATP aptamer with ATP results in the dissociation of the DNA duplex and the liberation of cDNA, which thereby causes the release of the intercalated DOX from DOX-Duplex through a structural transformation from the duplex to the aptamer/ATP complex (Figure 1b). The cationic protamine is employed to condense the anionic DNA scaffold to a complex (DOX-Complex), which has cell-penetrating and nucleus-targeting capabilities.
A liposomal membrane modified with a cell-penetrating peptide (CPP, R6H4) and having the fusogenic lipid composition of dioleoylphosphatidylethanolamine (DOPE) is coated on the core complex to obtain the DOX-Duplex-loaded fusogenic liposomes (DOX-FL), which have an acid-triggered fusogenic potential with the ATP-loaded liposomes (ATP-L) or endo-somes/lysosomes (endo-lysosomes).
As illustrated in Figure1c, after internalization by the tumor cells, DOX-FL and ATP-L are expected to be localized in the endo-lysosomes. In these intracellular acidic compartments, the membrane fusion of DOX-FL with ATP-L is induced due to the pH-sensitive fusogenic potential of DOPE enhanced by the R6H4 peptide, which leads to the exposure of DOX-Duplex to co-delivered ATP and subsequently triggers the release of intercalated DOX. Meanwhile, DOX-FL also possesses fusogenic activity with the endo-lysosomal membranes, thereby promoting endo-lysosomal escape and facilitating the transport of the released DOX to the cytosol, where it then specifically accumulates in the nuclei to eventually trigger cytotoxicity and apoptosis.
The intracellular trafficking profile of this co-delivery system was evaluated by CLSM (confocal laser scanning microscopy). Human breast cancer (MCF-7) cells were incubated with a mixture of DOX-FL and NBD-labeled ATP-L (NBD-ATP-L) for different time periods (Figure 4a). As the incubation time increased from 0.5 to 2 h, DOX-FL (red) and NBD-ATP-L (green) were effectively internalized by the cells and localized into the endo-lysosomes (blue), judged by the increased white pixels. The localization ratio between DOX and the endo-lysosomal marker LysoTracker increased from 29% to 80% (Figure 4b), which was an indicator for endo-lysosomal entrapment. After 2h incubation, the excess liposomes were removed and the cells were then incubated with fresh culture media for an additional 1, 2, or 4h. The separation in the signals of DOX, NBD, and LysoTracker was observed, determined by the decreased white and increased individual color pixels (Figure 4a).
The localization ratio of DOX with LysoTracker reduced to 24% after an additional 4h of incubation (Figure 4b), which suggested the efficient endo-lysosomal escape of DOX due to the membrane fusion between DOX-FL and endo-lysosomes along with the proton sponge effect of DOX-FL. In addition, the localization ratio of DOX with NBD decreased from 88% to 18%, and the co-delivery of DOX-FL with ATP-L showed the increased release of DOX in the cells compared with that of DOX-FL with Blank-L, indicating that a significant part of DOX-FL fused with ATP-L and activated the ATP-mediated release of the encapsulated DOX in the cells.
Furthermore, the released DOX was clearly visualized in the nuclei after an additional 2h of incubation, and increased considerably as the incubation time extended to 4h (Figure 4a), which implied the efficient nucleus-targeting of the released DOX.
We then applied a model (MCF-7 cancer xenograft nude mice) to evaluate the retention capacities of the liposome-based co-delivery system in tumor tissue. The signal of DOX delivered by DOX-FL in the frozen tumor sections was prominently higher than that of the DOX solution, which could still be observed even at 72h post-injection. In contrast, the relevant signal from samples associated with the DOX solution almost disappeared at 48h post-injection. It was validated that DOX-FL extended the tumor residence of the DOX molecule partially due to enhanced tumor penetration and cellular uptake upon R6H4 modification on the liposomal surface, while the DOX solution was subject to a more rapid clearance from the tumor tissue.
Next, the in vivo antitumor efficacy of the co-delivery system was investigated. After intratumoral injection, different DOX formulations significantly inhibited the tumor growth of the mice relative to that of mice injected with saline (Figure 5). DOX-FL with and without ATP-L both showed remarkably higher inhibition effects toward tumor growth than the DOX solution, which was due to the enhanced tumor retention capabilities of DOX-FL decorated with R6H4. Of note, DOX-FL co-delivered with ATP-L showed a noticeable difference in tumor-size inhibition compared to that of DOX-FL alone, indicating that the extrinsic ATP played an effective role in the DOX release. Additionally, the histologic images stained by the hematoxylin and eosin (HE) exhibited a massive cancer cell remission in the tumor tissue of the mice receiving DOX-FL with ATP-L.
Besides intratumoral injection, passively targeting-based co-delivery of DOX-FL with ATP-L by intravenous administration also showed promising tumor growth inhibition effects compared with that of the DOX solution and DOX-FL.
THE ATP COULD BE THE KEY
Enhanced Anticancer Efficacy by ATP-Mediated liposomial drug delivery, 2014.
Stimuli-responsive drug-delivery systems (DDSs) are playing an increasingly crucial role in a diverse spectrum of applications for disease treatment. The environmental stimuli involve external triggers, such as temperature, light, magnetic field, ultrasound, electric current_, radio waves_, and g-radiation_, as well as internal factors, such as pH, redox reactions, enzymatic expression, glucose levels, and the presence of reactive oxygen species. Nanoparticle(NP)-based DDSs including liposomes, polymeric NPs, and protein/DNA-based nanocarriers have been extensively investigated to respond to these signals for delivery of their cargoes in an on-demand fashion.
Adenosine-5’-triphosphate (ATP), the essential biogenic biomolecule for cellular energy metabolism and signaling, is highly present within the cells at a concentration range of 1-10 mm, much greater than that of ATP in the extracellular environment (<5mm). The distinct difference in the ATP levels between extracellular and intracellular milieu is the biological principle for the design of ATP-responsive carriers, which is recently attracting considerable interest. The existing ATP-responsive anticancer drug delivery methods are often limited by complicated formulation design and relatively low loading capacity of drugs. Some new carriers have been studied to overcome these limits.
DRUG DELIVERY WITH GRAPHENE STRUCTURE
ATP-responsive DNA-graphene hybrid nanoaggregates for anticancer drug delivery, 2015.
ATP-responsive anticancer drug delivery strategy utilizing DNA-graphene crosslinked hybrid nanoaggregates as carriers has provided many improvements. This nanoaggregate consists of graphene oxide (GO), two single-stranded DNA (ssDNA, denoted as DNA1 and DNA2) and ATP aptamer. The GO nanosheet is applied to carry doxorubicin (DOX), a model small-molecule anticancer drug. The single-stranded DNA1 and DNA2 together with the ATP aptamer serve as the linkers upon hybridization for controlled assembly of the DNA-GO nanoaggregates (DNA-GA). Both DNA1 and DNA2 are composed of “head” (target-specific sequence) and “tail” sequences (complementary to the target ATP aptamer and a repeated GT sequence).. DNA1 and DNA2 are separately added to the DOX-loaded GO (DOX/GO) solution to form the DOX-loaded DNA-GO complex (DOX/DNA-GC) via strong interactions including van der Waals forces, π-π stacking and hydrogen bond. When the ATP aptamer is added into the mixture of DOX/DNA1-GC and DOX/DNA2-GC (DOX/DNA12-GC), the hybridization of the ATP aptamer with both DNA1 and DNA2 results in the assembly of the GO nanosheets to form the layered-structural DOX-loaded DNA-GO nanoaggregates (DOX/DNA-GA). Such aggregates, with an increased average size and a decreased specific surface area toward the surrounding medium, can effectively inhibit DOX release from the GO nano sheets.
The ATP aptamer has been widely used for ATP detection based on its specific and stable binding to ATP. In the presence of ATP, the responsive formation of the ATP/ATP aptamer complex causes the dissociation of DOX/DNA-GA into DOX/DNA-GC that has a decreased size and an increased surface area exposing to the medium, which promotes the release of DOX in the environment with a high ATP concentration such as cytosol compared with that in the ATP-deficient extracellular fluid. This supports the development of a novel ATP-responsive platform for targeted on-demand delivery of anticancer drugs inside specific cells.
The intracellular distribution was visualized using CLSM (Fig. 5c). DOX and the stained nuclei displayed red and blue fluorescence, respectively.
After 2h of incubation, DOX/DNA-GA was endocytosed by the cancer cells and evenly distributed within the cells. As the incubation time was prolonged to 6h, DOX was efficiently released from GO, and the released DOX was specifically accumulated into the nuclei for subsequently inducing cytotoxicity, as observed by the magenta fluorescence.
The in vitro cytotoxicity of DOX/DNA-GA toward the cancer cells was evaluated using the MTT assay (Fig. 5d-e). DOX/DNA-GA presented an efficient and comparable cytotoxicity to DOX/DNA-GC against HeLa cells, indicating that the intracellular high ATP concentration resulted in the disintegration from DOX/DNA-GA to DOX/DNA-GC and therefore supported the parallel DOX release of DOX/DNA-GA with DOX/DNA-GC (Fig. 5d).
Nevertheless, the free DOX solution showed the highest toxicity to HeLa cells under the same condition due to the partial inefficient release of DOX from the GO surface. The cytotoxicity of DOX/DNA-GA significantly increased when the incubation time increased (Fig. 5e), suggesting that the sustained releasing DOX from DOX/DNA-GA allowed the enhanced cytotoxic effect toward the cancer cells.
APPLICATIONS USING NOT ONLY THE BIOCHEMISTRY
Drug Release from Electric-Field-Responsive Nanoparticle, 2012.
Another way that can demonstrate how much the science’s fields are nowadays more linked and more cooperative than ever. The scientists in this experiment tried a different way to provoke the release of the drugs in the specific tissue. They use nanoparticles reactive to a external DC electric field.
In summary, the researchers are working very hard trying to develop the next generations carriers that could one day revolve the strategies against many diseases, one upon all the cancer. Being able to make the carriers release in a very precise location the amount of drug would solve many problems by focusing in the intra-cellular action. The straightforward formulation design, high loading capacity of drugs and capability of site-specifically promoting drug release are just some of the pros.