Design of A Nanoparticle Of A DNA Plasmid For Intracellular Delivery
Cover letter
According to modern research, cell penetrating peptides (CPP) entirely consists of fundamental amino acids resides that has the potential of crossing cytoplasmic membranes for the purpose of delivering important biological molecules into cells. Despite that, the CPP or the cargo entrapment contained in the endosome has the capability of limiting biomedical utility once the cargos are being destroyed in the prevailing acidic environment. On the other hand, the protein transduction of the novel cell penetrating peptides (CPP) consists of the nona-arginine (IR9) and the INF7 fusion peptide that is used for the purpose of enhancing delivery of the vital molecules in cells. What this implies is the fact that the IR9 has the ability of interacting with DNAs and the quantum dots (QDs) for the purpose of forming stable IR9/QD as well as the IR9/DNA complexes. These complexes are vital because they have the propensity of entering human A549 cells.
In connection with that, zeta-potentials are ultimately the best means of predicting transduction efficiency as compared to gel shifting analysis. What this implies is the general significance of the electrostatic interaction of the cell penetrating peptides (CPP) or the cargo complexes with the human plasma membranes. According to the mechanistic research, IR9/DNA and IR9/QD complexes have the capability of entering human cells through the process termed as endocytosis. Furthermore, the IR9/DNA and IR9/QD cannot be cytotoxic when their concentration falls. This implies that IR9 is the ultimate component that can be used for the purpose of carrying genes as well as drugs for various biomedical applications.
Specific aim
The aim of this research will entail determining whether the cell penetrating peptides has the potential of enhancing molecular activities when various molecules are tarnsfred in and out of the cells.
Introduction
The cell penetrating peptides (CPPs) can be categorized into various classes namely; chimeric, synthetic, and protein-derived. For example, penetratin and Tat, the first two categories of CPPs are considered to be protein-derived. Amphipathic peptide and nona-arginine (R9) do not possess natural proteins hence making them to one of the synthetic molecules. On the other hand, the members of the chimeric family have the possibility of incorporating the main functions of the natural proteins, for instance, that of transportan and Pep-1 respectively. The division of each CPPs family is based on their sequence or original characteristics (Langel, 2019).
Since CPPs has the potential of delivering important biological material into the cell membrane, it is perceived to an important tool in therapeutics. Some of the modern clinical experiments that have been conducted have been aimed at using CPPs in delivering macromolecular medicinal conjugates into patients suffering from multiple ailments (Li et al., 2017). The reason for that is because it has the capability of carrying with it multiple spectrums of cargo molecules, for instance, nucleic acids, cytotoxic drugs, proteins, liposomes, peptide nucleic acids, and so on. Accordingly, research indicates that the general internalization of the kinetics of CPPs is something that is rapid. Since the CPPs have been found to non-toxic to the majority of the cell, its safety can ultimately be analyzed using metabolic analysis (Yuan, 2011).
As stated above, quantum dots (QDs) are some of the inorganic semiconductor nanocrystals that have various excitation molecules taking into account their size and emission properties. Despite that, it should be understood that they do not possess the capacity of penetrating cells. The aggregation mainly occurs during internalization. To have the potential of overcoming these limitations, the modification of their surfaces using noncovalent or covalent linkages with the CPPs is essential. The facilitation of QDs using CPPs is the one that has the potential of reducing the nonspecific absorption as well as other associated side effects (Betty et al., 2013). Regardless of that, research indicates that QDs are venerable to sequestration and entrapment by lysosomes or endosomes in cells (Lin et al., 2009).
Accordingly, endosomoltic agents and transduction enhancers can also be used for the purpose of improving the transduction efficiency of CPP as well as to aid in overcoming lysosomal or endosomal entrapment. The majority of the enhancers used have the potential of increasing the general hydrophobicity of the CPPs as well as increasing cell membrane permeability. On the other hand, chloroquine, for instance, is one of the lysosomotrpic agents that aid in preventing lysosomal trapping. It should be understood that endocytosis is the ultimate mechanisms that are used for the purpose of enhancing the cellular intake of CPPs. Moreover, the quick discharge from the endocytic vesicles to the cytosol is fundamental in preserving all the biological activities of the molecules or cargos. Some of the endosome-desruptive peptides also termed as lysosomotrophic peptides can also be obtained from bacterial or viral toxins. The importance of these peptides is that they aid in triggering the acidification of endosomal which in return enables the cargos to move and enter the cytosol.
Overall aim
The essence of this research will entail creating a chimeric IR9 CPP that contains both designated IR9 and INF7 fusion peptide. The next step will entail assessing the main IR9 transduction process through the transportation of DNAs and QDs by the cell mebrane. The research will further investigate the mechanisms used by CPP to transport DNAs and QDs into cell membrane. In order to achieve these objectives, IR9 will be synthesized before examining IR9or DNA, IR9 or QD, and IR9 by the cell membrane suing flow cytometry and live cell imaging. Zeta-potential analyzer will also be used to aid in illustrating the relationship the characterization that exists between electrostatic interactions, charging state, and the transduction efficiency of IR9. Furthermore, to be in the position of elucidating the IR9/cargo complexes and IR9 uptake mechanisms, pharmacological and physical inhibitors will be taken into consideration to aid in blocking certain endoctytic pathways. The results obtained are the ones that will be used for the purpose of analyzing the prevailing cytotoxicity mechanisms of IR9/cargo complexes and IR9 uptake (Lin et al., 2009).
Governing hypothesis: The cell penetrating peptides (CPPs) has the potential of penetrating the cell membrane as well as transporting various biological molecules
Specific aim 1: The cell penetrating peptides will be investigated to aid in determining the manner in which it assists in the transportation of cellular molecules taking into account its molecular properties.
Background
A cell membrane is perceived to be a barrier that aid protecting cells from the entry of external materials through monitoring the inflow and outflow of such molecules. The presence of the cytoplasmic membranes also aid in mediating various essential processes, for instance, nutrient intake, cell wall biogenesis, secretion, cellular morphogenesis, and environmental sensing. What this implies is the fact that the significance of the plasma membrane ultimately takes into account the manner in which the majority of the pharmaceutical components end up targeting its components. Ideally, the transportation of various exogenous molecules across the plasma membrane is a mechanism that is ultimately influenced by protein, glycolipid, phospholipid, and cholesterol components (Li et al., 2017). The permeability of the plasma membrane ultimately depends on certain material transporters as well as the polarity and the size of the material being transported. When certain material transporters are not present, small hydrophobic molecules will only be permitted to pass into the cell by the membrane. It should be understood that large biological macromolecules and hydrophilic drugs, including RNAs, DNAs, and proteins cannot be allowed to pass through the cell membrane freely (Betty et al., 2013).
Nevertheless, the aim of this research will entail illustrating how the composition of the protein transduction domains or the cell penetrating peptides (CPP) consisting of small peptides that has the potential to traverse the cell membrane and in return deliver various materials into the cell. Normally, the truncated Tat proteins enable the movement of the peptides into the cell membrane as it penetrates it before they accumulate the cell nuclei. This implies that one of the main features of the CPPs entail its capability of transporting various essential molecules into the cell. Cationic, hydrophobic, and ampipathic peptides are the main constitutes of the CPPs (Liu et al., 2013).
Planned research
This research will take into account the investigation of the properties of the cell penetrating peptides in conjunction with the manner in which quantum dots end up enhancing its cellular activities. The same mechanism will take into consideration some of the difficulties that are encountered during the delivery of biological molecules into the cells.
Specific aim 2
The aim of this research will entail determining whether the cell penetrating peptides has the capacity of enhancing the transportation of various biological components into living cells.
Background
The CPPs (cell penetrating peptides) has the potential of penetrating cells without the production of proteins from prokaryotes. Due to the fact that IR9 is one of the chimeric molecules that were obtained as a result of the fusion of the fusogenic peptide INF7 and synthetic nona-arginine, it was possible to understand its penetration mechanisms. It was found out that IR9 has the potential of noncovalently interacting with DNAs and QDs to form stable complexes aid in delivering them into human A549 cell membrane.
Moreover, a high correlation was obtained between the transduction efficiency of protein and zeta-potential of the APCs/DNA complexes. On the other hand, the electrostatic interactions of the cargo or the IR9 complexes with the plasma membrane have been realized to play a crucial role in regulating cellular internalization. As a result of that, it is evident that endocytosis is the ultimate means that can be used for the purpose of enhancing the uptake of IR9 complexes (Ohshima & Makino, 2014). These characteristic indicates that IR9 can be used as the ultimate tool for studying various biological processes, for instance delivery vectors and gene expression in numerous biomedical applications
Planned research
In order to understand the biological applications of CPPs, human bronchoalveolar carcinoma A549 cells supplemented with 10 percent bovin serum. Living cells were examined using propidium iodine stain. Phosphate buffered saline was used to wash the living cells. The culture was transferred to RPMI 1640 that before adding a controlled percent of serum during the incubation process. Quantum Dots (QDs) and CPPs were measured so as to determine their emission peak wavelengths.
IR9 peptide was mixed with quantum dots (QDs) so as to prepare QD/IR9 complexes at different molecular ratios. Electrophoresis was used for the purpose of analyzing QD/1R9 on agarose gel. In order to prepare DNA/IR9 complexes, an equal amount of IR9 was mixed with pEGFP-N1plasmid to encode the enhanced green fluorescent protein (EGFP) reporter gene. After duration of two hour incubation, the DNA/IR9 mixtures were also analyzed using the electrophoresis process. Images were captured and analyzed
The noncovalent quntum dots transduction was carried out using different amount of IR9-FITC (fluorescein isothiocyanate) peptide. The mixture was incubated with the A549 cells before being analyzed using the flow cytometry mechanism. The cells that were initially treated using FITC, PBS, or non-CPP were used as the negative controls. During the kinetic study of transductions, different amount of IR9-FITC was added to the cells at different time but maintained at the same temperature. So as to be in the position of determining the colocalization of the sub-cellular mixture of the IR9-FITC, organelle fluorescents was used to aid in visualizing lysosomes and the cell nuclei respectively.
In order to foster the transduction of the noncovalent IR9/cargo complexes, quantum dots (QDs) was mixed with IR9 peptide at different molecular ratio. After that, the QD/IR9 complexes which were initially prepared were incubated for one hour at 37o C. So as to study the cells’ transduction kinetics, all the materials were prepared using IR9/QD complexes that were initially prepared at different molecular ratios. During the same experiment, the cells with high reactivity ratios were analyzed using a flow cytometer or a confocal microscope. To analyze the dissociation of the quantum dots (QDs) from the cell penetrating peptides (CPPs), IR9-FITC was used for the purpose of incubating the cells for at least 24 hours. After incubation, the complexes was removed and then stained before observing them using the confocal microscope. Chloroquine was added to the mixture so as to determine the cells’ lysosomal escape.
Pharmacological and physical endocytic modulators were used for the purpose of evaluating the effect of endocytosis in intricate transduction. IR9/DNA, IR9/QD, or IR9-FITC was incubated together with the human cells at 5o C. In order to be in the position of analyzing the significance of macropinocytosis in complex cell transductions, all the cells were mixed with cytochalasin D before uptake was determined (Dietz et al., 2009).
Expected outcomes
A graph showing the percentage relative shift of IR9/QD against IR9/QD ratio
Y-axis
120
100
Percentage 80
Relative shift 60
40
20
20 40 60 80 100 120 X-axis
IR9/QD ratio
From the graph above, the poor intracellular tracking trafficking as well as the release of endosomal molecules is the main factors that have the potential of reducing the transduction efficiency of CPP proteins that is mediated by the existing endocytic pathways. From the information gathered, it is evident that endosomal entrapment has the potential of inducing enzymatic degradation of the cell penetrating peptides (CPPs) as well as their cargoes. The incorporation of INF7 and HA2 peptides into the (cell penetrating peptides cell penetrating peptides is the one that can aid in overcoming macropisomes or endosomes entrapments. The reason for that is because they have the potential of inducing perturbation of the vesicle membranes (Langel, 2019).
The significance of the endosomolytic HA2 tag is that has the potential of increasing cellular uptake, accelerating endosomal escape, as well as promoting the cytosolic distribution of the andocytosed cell penetrating peptides (CPPs) that contain RFPs in the human A549 cell membranes. Ideally, it is evident that the comparative IR9-FITC with R9-HA2 was the potential of determining the one that has the ability of increasing transduction efficiency. Despite that it is evident that both of them have the ability of reducing cell viability taking into account their concentrations (Huang et al., 2005).
On the other hand, the potential of the membrane is the one that assists in the internalization of the arginine-rich cell penetrating peptides towards the cell membrane. What this implies is the fact that the change of state of the cargo or the IR9 complexes is the one that has the capacity of influencing the efficiency of the cellular internalization mechanisms of CPP. Ideally, the transduction of the proteins that are mediated by the cell penetrating peptides can be illustrated using three steps (Düzgüneş, 2012). The first one entail binding the cell membrane, the second one is its capability of penetrating cell, and the third one entail the release of specific or cytoplasmic organelles.
A graph showing the cell transduction efficiency against time
Y-axis
120
100
Transduction 80
Efficiency 60
40
20
1 2 3 4 5 6 7 8 9 10 X-axis
Time (minutes)
Cellular uptake is a mechanism that is ultimately enhanced through the electrostatic interaction between negatively charged plasma cell membrane and CPP or cargo complexes. For example, surface charge has been realized as being the main determinant when it comes to the flow of nanoparticles end up impacting the whole cellular processes. Some of the cellular processes that are impacted include mitochondrial functions, cell morphology, cytotoxicity, and the level of intracellular calcium. Since both the negatively and positively charged particles are ultimately cytotoxic, it implies that the negative one are always more toxic as compared to the positive ones. Zeta-potential always varies greatly taking into consideration the mechanism of producing and treating them, their nanoparticle size, the structure of their surface, as well as other associated properties (Baharvand & Aghdami, 2014). On the other hand, electrostatic properties of the cell membrane are used for the purpose governing the general interactions of the cargo complexes or the cell penetrating peptides (CPPs) or APCs. In connection with that, plasma membranes that are negatively charged have been realized to aid in determining transduction efficiency.
A graph showing the percentage cell viability of IR9/QD against IR9/QD cytotoxicity
Y-axis
120
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A |
100
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C |
Percentage 80
Cell viability 60
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B |
40
20
1 3 6 9 12 X-axis
N/P and IR9/QD cytotoxicity
KEY
A =Positive control
B= Negative control
C= Deoxyribonucleic acid (DNA) only
The understanding that was obtained through the cellular internalization of the use of the cell penetrating peptides illuminates the fact that it has the potential of utilizing several pathways when it comes to cellular entry. The two major pathways that are used for the cellular intake of the cell penetrating peptides are mainly the nonendocytic and endocytic pathways. Classical endocytosis is ultimately an energy-depended pathway of the CD8+ T cells (Donnelly et al., 2005). On the other hand, the nonendocytic route also termed as the pore-opening, direct penetration, or direct membrane translocation, is perceived to one of the rapid as well as the energy independent pathways. Accordingly, Tat, R9, and antennapedia have been realized to have the capacity of using at least main endocytic pathways. These pathways include, lipid/caveolae-raft mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis. From the information collected, it was evident that endocytosis is the ultimate means that is used for the purpose of enhancing cellular intake of the IR9 as well as the IR9 cargo complexes. In connection with that, the main pathways that are used for the purpose of enhancing the internalization of QDs mainly rely on the conjugated molecular carriers or peptides (Devarajan et al., 2015). What has the potential of influencing the pathways and the cellular uptake of the cell penetrating peptides include the cargo characteristics, APCs/CPP complexing methods, CPP concentration, CPP properties, composition of cell membrane, serum concentration, and transduction period.
In accordance with that, it is evident that surface charge is one of the factors that can aid in determining the manner in which gold nanoparticles end up impacting cellular processes, for instance cytotoxicity, levels of intracellular calcium, mitochondrial functions, and cell morphology. Regardless of the fact that both the negatively and positively charged nanoparticles are cytotoxic, the negatively charged gold particles have been found to be more toxic as compared to the positive ones. What was realized from the data collected is the fact that the electropositive zeta-potential of the cargo/IR9 complexes has the potential of improving its transduction efficiency. Electropositivity values obtained used as the limit for separating high-charged surface from the low-charged surface is the one that determines molecular suspension stability (Dietz et al., 2009). Conversely, the electrostatic properties that govern APCs/CPP complex correlation with the negatively charged cell membranes are the main factors that aid in determining the transduction efficiency of the transported molecules.
Although research indicates that cellular internalization of the cell penetrating peptides is a complex mechanism, it is evident that CPP utilizes more than two pathways when it comes to cellular entry. Moreover, the QDs internalization pathways that are involved mainly rely on their carriers or conjugated peptides. Some of the factors that have the potential of influencing cellular intake efficiency and the pathways for CPPs include its complexing method, serum concentration, CPP concentration and properties, transduction duration, cargo characteristics, sand so on (Lin et al., 2009).
Perspectives
From the information collected above, what this implies is the fact that cell penetrating peptides (CPP) and IR9 interacts noncovalently with DNAs or QDs for the purpose of forming stable complexes that has the capability of delivering them into the cells. Accordingly, the electrostatic properties of the cargo/IR9 components with the cells have been found to play key role cell internalization. IR9 and IR9/cargo complexes have the potential of entering the cell membrane though the process termed as endocytosis. In connection with that, IR9 consists of INF7 fusogenic domains that assist in promoting the general release of cargo or IR9 complexes from endosomes. Since IR9 is somehow nontoxic, it means that it can be utilized as the main carrier of medical cargoes in various biomedical applications.
In connection with that, it is evident that membrane potential plays a crucial role when it comes to the internalization of arginine-rich cell penetrating peptides (CPP) into the cell membrane. The charge state of the cargo/IR9 complexes is the one that has the potential of influencing the efficiency of the cell penetrating peptides during cell internalization. The protein transduction of the cell penetrating peptides (CPP) or APCs mediated cargo transportation takes three stapes. The first one takes into account the binding of the cell membranes, the second one entail its general penetration into the cell membrane, and the last one involves their release into the cytoplasm or certain organelles. Cellular uptake, as the first step of the properties of the cell penetrating peptides is something that that is enhanced by the electrostatic interactions that exist between the negatively charged plasma membranes and the cargo or CPP complexes.
Weekly work plan (grant chart)
|
|
TASK |
PREDECESSORS |
TIMELINE |
|
1 |
CPPs materials acquisition |
1 (CPPs material preparation) |
1 day |
|
2 |
Reacting material with required supplements |
2 (investigating the chemical properties of the cells that were initially prepared |
2 days |
|
3 |
Leaving the mixture to react |
3 (conforming the chemical nature of the material prepared after reacting) |
1 day |
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4 |
Analyzing the cytoxocity properties of the material prepared |
3 (determining the penetration properties of cellular molecules) |
1 day |
Research and economic impact
Some of the factors stated above have the potential of influencing cellular intake efficiency and the pathways for CPPs include its complexing method, serum concentration, CPPs or APCs concentration and properties, transduction duration, cargo characteristics, sand so on. The electrostatic properties that govern cargo/CPP complex correlation with the negatively charged cell membranes are the main factors that aid in determining the transduction efficiency of the transported molecules. With the negatively and the positively charged nanoparticles, research indicates that the negatively charged gold particles have been found to be more toxic as compared to the positive ones.
In accordance with that, it should be understood that protein transduction of the cell penetrating peptides (CPPs) or APCs mediated cargo transportation takes three stapes. The first one involves the binding of the cell membranes. The second one deals with the general penetration into the cell membrane. The last one involves the release of molecules into the cytoplasm or certain organelles. Therefore, what this implies is the fact that the cellular uptake, as the first step of the properties of the cell penetrating peptides is something that that is fostered by the electrostatic interactions that exist between the negatively and the positively charged plasma membranes and the cargo or CPPs or APCs complexes. Conversely, the understanding obtained through the cellular internalization highlights the fact that it has the possibility to utilize various pathways when it comes to cellular entry.
Suitability of application
The electrostatic interaction of the cargo or the IR9/DNA components was found to have the potential of enhancing cell internalization. This, therefore, suggests that endocytosis is the ultimate means that can be used for the purpose of enhancing the uptake of IR9 complexes. These characteristic indicates that IR9/DNA complexes can be used as the basic tool for studying various biological processes, for instance the expression of genes and delivery vectors. Accordingly, what this implies is the fact that endocytosis is the ultimate mechanisms that are used for the purpose of enhancing the cellular intake of CPPs.
Although CPPs does not have the capability of penetrating cells, such an aggregation ultimately occurs during cell internalization. So as to overcome these limitations, noncovalent or covalent linkages with the cell penetrating peptides (CPPs) is used. Therefore, what this implies is the fact that CPPs are capable of transporting vital biological materials into the cell membrane for medicinal purposes. From the research conducted, it is possible to deliver macromolecular substances into the cells of the patients suffering from various diseases. It is the capability of CPPs to carry with it multiple spectrums of cargo molecules that makes that possible.
References
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