Subscriber access provided by ECU Libraries
B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules
Improved Intracellular Delivery of Polyarginine Peptides with Cargoes Juanmei Hu, Yimin Lou, and Feng-min Wu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10483 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Improved Intracellular Delivery of Polyarginine Peptides with Cargoes Juanmei Hu, Yimin Lou, Fengmin Wu* Key Laboratory of Optical Field Manipulation & Center for Optoelectronics Materials and Devices of Zhejiang Province, Department of Physics, Zhejiang Sci-Tech University, Hangzhou, 310018, China Abstract: Complementary to endocytosis, cell penetrating peptides (CPPs) at high concentration can penetrate cell membrane in a direct way, which further makes CPPs popular candidates for delivering therapeutic or diagnostic agents. Although featured as rapid uptake, the translocation efficiency and potential toxicity of the direct penetration are usually affected by cargos, which is still unclear. Here, using coarse grained molecular dynamics simulations we show that the polyarginine (R8) peptides penetrate the membrane through a water pore in the membrane and the transmembrane efficiency is improved by conjugating to small nanoparticles (NP) with proper linkers. It can be attributed to both the extension of water pore’s lifetime by the nanoparticles and outward diffusion of negative lipids in the asymmetry membrane, which induces the surrounding R8-NP conjugates to the water pore before it is closed. The translocation efficiency is closely related to the length of the linkers and it gets the maximum value when the length of linkers is around half of the membrane thickness. Overlong linkers not only decrease the transmembrane efficiency due to the blockage of nanoparticles in the water pore, but also may cause cytotoxicity due to the unclosed water pore. The results provide insights into the internalization of cell penetrating peptides and facilitate the design of cell penetrating peptide and drug conjugates with high efficiency and low toxicity. INTRODUCTION Cell penetrating peptides (CPPs) have become promising candidates for the delivery of therapeutic or diagnostic agents into cells due to the efficient membrane penetration ability.1,2 The cargoes are versatile, including nucleic acids,3 active
*
[email protected] ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
proteins,4 quantum dots5 and nanoparticles.6 Therefore, efforts are being invested to explore the transmembrane mechanism, as well as to design and synthesize more efficient and less toxic CPPs.7-9 Recently, increasing experiments and simulations support the view that direct translocation becomes dominant when the concentration of CPPs is high.10-12 This translocation mechanism indicates that the positively charged amino acids, such as arginines, interact strongly with phosphates groups on both sides of the membrane, which distorts the membrane structure and initiate a hydrophilic water pore in the membrane.13,14 Then part of the CPPs translocate to the inner leaflet of the membrane through the water pore. According to this mechanism, numerous efforts have been exerted to looking for new CPPs or other conditions that make the membrane easy to produce water pores. The works include the change of amino acid types and sequence of the CPPs,15-17 as well as their second structures18-22 and aggregation degree.23-25 In addition, the composition of the cell membrane,26-28 its stress and the external electric field29-31 have also been studied. Studies have demonstrated that the transmembrane delivery is not only dependent on the properties of CPPs, but also sensitive to the cargos and their binding ways.32-34 Therefore, reforming the CPP-cargo conjugates by modifying the properties of the cargos and their conjugation modes are more straight ways to design and synthesize drug delivery systems with high translocation efficiency and low side effects. In experiments, CPP and cargo are usually covalently bonded by a linkage or non-covalently assembled by intermolecular interactions.6,35-38 In the former case, the most common way is tethering the a cargo or drug to the CPP molecule through a linker, such as hydrazone bond, or disulfide bond which is cleavable under suitable conditions.39-41 In such cases, the transmembrane mechanism may not only be affected by the cargos, but also influenced by the linkers.42 However, the cellular selectivity and biodegradability were usually concerned in choosing linkers, while their effects on the drug transport mechanism and transport efficiency were neglected.43-47 Therefore, study the effect of cargoes with proper linkers on the
ACS Paragon Plus Environment
Page 2 of 26
Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
transmembrane mechanism of CPPs is important in designing new CPP-cargo conjugates with improved membrane translocation abilities. In this study, the membrane translocation of polyarginine (R8) peptide and small hydrophilic nanoparticle (NP) conjugate that are covalently bonded by a hydrophilic linker is investigated by coarse-grained molecular dynamics simulations.48 Results show that the transmembrane efficiency of R8 peptides is improved after carrying small hydrophilic nanoparticles. In addition, the membrane translocation of R8-NP conjugates is affected by the length of the linkers. Overlong linkers not only decrease the transmembrane efficiency but also may cause cytotoxicity. The mechanism is discussed in detail. Our results provide insights in designing CPP-cargo conjugates with high transmembrane efficiency and low toxicity. MODEL AND METHODS
Figure 1. The coarse grained structure of (a) DMPC and DMPS lipid, (b) asymmetry lipid bilayer, (c) R8 peptide, and (d) R8-NP conjugate. The nanoparticle is covalently bonded to one terminal of the backbone of R8 peptide with a hydrophilic linker (the pink beads). The types of the beads are described beside.
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 26
The coarse-grained molecular dynamics simulations were carried out with Martini force field (version 2.1).49 In Martini force field, four heavy atoms are represented by one bead which is defined as one of the four types: charged (Q), polar (P), nonpolar (N), apolar (C), as shown in Figure 1. It neglects the atomic degree of freedom and has been proved feasible to simulate biological systems, such as membrane, proteins and DNA.50,51 In our simulation, the membrane is composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC)
and
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) lipids. For DMPC lipid, the choline and phosphate are represented by charged beads Q0 and Qɑ, which contains one positive charge and one negative charge, respectively. The glycerol ester is represented by two nonpolar beads Nɑ. Each of the two hydrophobic chains is represented by three apolar beads C1. For DMPS lipid, the charged bead Q0 in DMPC is changed to polar bead P5. Each arginine is mapped into three beads: one polar backbone bead (P5) and two side-chain beads (N0 and Qd). The R8 peptide is composed of eight arginines connected by the backbone beads with an extended structure. The nanoparticle with a diameter of about 2 nm is composed of twenty beads in fcc structure. The nanoparticle is tethered to one end of the backbone of R8 peptide by a linker with beads number from zero to eight. To avoid aggregation under physiological conditions, the beads in the nanoparticle and linker are treated as P1.52 During the whole simulation, the nanoparticle moves as a rigid body. The detailed parameters for the interactions of lipid, R8 peptide, nanoparticle and linker are given in Table S1 in the Supporting Information. For most eukaryotic cell, the lipid composition across cell membrane is asymmetric, which has been proven to be involved in a variety of membrane-based functions, such as translocation and signal transmission.53,54 Simulation studies have also confirmed the importance of asymmetric membrane on the translocation of cell penetrating peptides.30,55 In this study, an asymmetric membrane model is used. The outer leaflet of the membrane is composed of 324 DMPC lipids while the inner leaflet is composed of 162 DMPC lipids and 162 DMPS lipids. It is similar to the human
ACS Paragon Plus Environment
Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
erythrocyte membrane, in which the negatively charged phosphatidylserine (PS) is predominately located on the cytosolic leaflet while the electrically neutral phosphatidycholine (PC) is located on the extracellular leaflet. Beside, 36000 standard water beads are introduced to the simulation system with ten percent of antifreeze beads. To neutralize the whole system, sodium ions are added in the simulation system. Recent studies have shown that the energy barrier for the formation of hydrophilic water pore in the membrane is concentration dependent due to the electrostatic interactions and cooperation effect of arginine-rich peptides.56 Figure S2 shows that the membrane enlarges after adsorption of R8-NP conjugates and its maximum size is dependent on the concentration of the R8-NP conjugates. No penetration is observed during the simulations when there are nine R8-NP conjugates on the membrane surface, as shown in Figure S3. Therefore, the critical concentration to induce the water pore in our system is ten R8 peptides. At first, the membrane system with nine R8 peptides adsorbed on the out leaflet surface was equilibrated for 40 ns. In such equilibrated state, the simulation box is 15.5 nm×15.5 nm×24.0 nm. Then, different numbers of R8 peptides or R8-NP conjugates were placed above the membrane, and the corresponding numbers of counterions were removed. Before further simulation, R8 peptides or R8-NP conjugates, water molecules and the counterions were equilibrated by 20 ns with the membrane fixed. After that, 1000 ns simulations were carried out to study the translocation process. Each case was simulated more than six times. The simulations were performed by the LAMMPS package (8 March 2018).57 NPT ensemble with a constant temperature of 323 K and a constant pressure of one atmosphere was used. The temperature of membrane and the other part were controlled by Berendsen thermostat independently. The pressure in x and y direction was coupled and that in z direction was controlled separately with Berendsen pressure method. The van der waals interactions were described by Lennard Jones potential which was cut at a distance of 1.2 nm. To reduce the cutoff noise, it smoothly
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
switched to zero between 0.9 nm and 1.2 nm. The coulombic interactions were cut at 1.2 nm and the long range part were calculated using PPPM method with the accuracy of 1.0 e-6. Studies have shown that pore formation is observed in the dendrimers and lipid bilayer systems when long range electrostatic interactions are concerned without significant effect on the membrane properties[30]. Relative dielectric constant of 15 and time step of 20 fs which corresponds to the effective time step of 80 fs were used. RESULTS AND DISCUSSION Direct membrane translocation of R8-NP conjugates
Figure 2. Direct membrane translocation of R8 peptides and R8-NP conjugates. Top view and back view of typical snapshots of (a) the R8 peptides and (b) the R8-NP conjugates in asymmetric membrane at 0 ns and 1000 ns. Ions and water are omitted for clarity. The distance between the mass center of CPPs and membrane as a function of simulation time for (c) the R8 peptides and (d) the R8-NP conjugates along transmembrane direction at the first 240 ns. A series of molecular dynamics simulations were performed to study the cargos
ACS Paragon Plus Environment
Page 6 of 26
Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
on the membrane translocation of cell penetrating peptides. Here, the membrane penetration of CPPs without (R8) and with nanoparticles (R8-NP) were both investigated at the concentration of twelve CPPs. For the R8-NP conjugate, a hydrophilic linker composed of four beads (about 2.43 nm which is comparable to half of the membrane thickness) was used to connect the R8 peptide and nanoparticle, as shown in Figure 1 (d). Snapshots and the evolution process of the membrane translocation of R8 peptides and R8-NP conjugates are shown in Figure 2. At first, twelve R8 peptides and twelve R8-NP conjugates were separately adsorbed on the outer leaflet of the membrane randomly. At equilibrate, three R8 peptides passed through the membrane and adsorbed on the inner leaflet at the end of simulation, as shown in Figures 2 (a). Differently, four R8 peptides penetrated the membrane when the R8 peptides were conjugated to nanoparticles, as shown in Figures 2 (b). Similar results were obtained by multiple simulations. It means that the intracellular delivery of R8 peptides can be improved by carrying hydrophilic nanoparticles. The detailed translocation processes are shown in the center of mass (COM) distance between the CPPs and membrane along transmembrane direction (z-direction) over time in Figures 2 (c) and (d). Combined with the translocation of R8 peptides in animation S1 and the z-distance variation in Figures 2 (c), we can see that the three R8 peptides do not interfere with each other during the membrane translocation. All the three R8 peptides penetrate to the inner leaflet quickly once the pore forms, which is consistent with previous findings [Error! Bookmark not defined.]. In contrast, the penetration of the R8-NP conjugates was much slower and the translocation processes of all the four R8-NP conjugates can be divided into two stages. The translocation mechanism of R8-NP conjugates was investigated by tracking the detailed penetration processes of twelve R8-NP conjugates in the asymmetric membrane, as shown in figure 3(a). The continuous translocation is given in the animation S2. For clarity, the penetration process at the first 120 ns was plotted and the side views were magnified. We can see that the translocation is mediated by a hydrophilic water pore in the membrane with the lipid heads around the pore and the
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
lipid tails buried in the inner of the bilayer. At 18.4 ns, the first R8 peptide arrived at the inner leaflet surface with the nanoparticle in the pore. At 33.6 ns, the second R8-NP conjugate reached the water pore and performed a similar direct translocation before the total translocation of the first R8-NP conjugate. Subsequently, the fourth and tenth R8-NP conjugates translocated across the membrane through the water pore at 64 ns and 96 ns, respectively. In this simulation, the four R8-NP conjugates penetrated the membrane through the same water pore and the water pore did not close until the total translocation of four R8-NP conjugates at 114 ns. In figure 3 (b), the time evolution of COM distance in z-direction between R8 peptides and membrane, as well as the nanoparticles and membrane were plotted separately. It shows that the nanoparticles always lag behind the R8 peptides during the translocation. Due to the electrostatic attractions between the R8 peptides and the DMPS lipids in the inner leaflet, R8 peptides translocate to the inner leaflet surface directly once it reaches the water pore. At the same time, the nanoparticles are dragged to the water pore. When the R8 peptides reach the inner leaflet surface, the dragging force decreases and nanoparticles will get to cytoplasm by thermo motion. That’s why the translocation process of the R8-NP conjugates has two stages. When the R8 of the subsequent R8-NP conjugate reaches the water pore and passes through the membrane, it will promote the translocation of the former NP. The whole translocation process of the R8-NP conjugates shows perfect sense of rhythm. The water pore will not close until one R8-NP conjugate have completely penetrated the membrane and the resting R8-NP conjugates have not yet reached the pore. Contrarily, the membrane translocations of R8 peptides in Figure S1 and animation S1 show that the water pores close immediately once one R8 peptide penetrates the membrane. In order to explain the different translocation between the R8 peptides and the R8-NP conjugates, the lifetime of the water pore was calculated. The lifetime is defined as the duration of water pore for penetrating only one R8 peptide or one R8-NP conjugate. The lifetime is counted when the nearest distance between the water molecules inside and outside the membrane was less than 1 nm. From statistical results, the lifetime of the water pore for the translocation of one R8-NP conjugate
ACS Paragon Plus Environment
Page 8 of 26
Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
with a linker of four beads is around 16.8 ns, while that of R8 peptides without carrying cargoes is only 6.8 ns. It is consistent with the current observations that duration of the transient water pore extends once larger or more molecules access the pore simultaneously.58,59
Figure 3. Translocation process of twelve R8-NP conjugates through the asymmetric membrane with the linker of four hydrophilic beads. (a) Time sequence of snapshots of the translocation process of R8-NP conjugates (top view, magnified side view and back view). (b) The time evolution of center of mass distance between R8 peptides and membrane (solid lines), and nanoparticles and membrane (dotted lines) of the four R8-NP conjugates that penetrated the membrane. (c) The number of outward DMPS and inward DMPS as a function of simulation time, insert is the schematic penetration of R8-NP conjugates.
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
To further investigate the transmembrane efficiency of R8-NP conjugates, the distribution of DMPS during the translocation process is analyzed. Results in Figure S4 show that flip-flop of lipids can be neglected without water pore in the membrane. Figure 3 (c) is the state of DMPS as a function of simulation time. For simplicity, the DMPS is defined as outward if the choline in one lipid is above the tail of the hydrophobic chains. Otherwise, it is defined as inward. Initially, all the DMPS lipids were placed in the inner leaflet. We can see that the number of outward DMPS has a sharp increase once the water pore appeared at around 12 ns and it increases continuously until the water pore closed at 114 ns. The directional diffusion of DMPS is similar to the ion current through the water pore [Error! Bookmark not defined.]. Based on the direct translocation mechanism, the water pore is mainly induced by the strong electrostatic interactions between the positively charged CPPs and the asymmetry membrane or anion in the cytoplasm [Error! Bookmark not defined.]. After translocation, the electrostatic interactions are strongly diminished and the water pore closed. Therefore, the translocation of CPPs is always accompanied by the outward diffusion of DMPS, which is similar to the outward ion current through the pore. As DMPS lipids diffuse out of the water pore, the remaining R8 peptides on the outer leaflet of the membrane will be induced to the water pore by the strong electrostatic attractions. If another R8-NP conjugate arrives at the water pore before the water pore closes, it will translocate across the membrane in a same way. The subsequent R8-NP conjugate further extends the lifetime of the water pore and more R8-NP conjugates can be induced to the water pore. That’s why the translocation efficiency of R8 peptides is improved after carrying hydrophilic nanoparticles with proper linkers. The schematic translocation of R8-NP conjugates is given in the insertion in figure 3(c). Moreover, the water pore can move towards nearby R8 peptides, which further promotes the penetration, see the animation in the supporting information (S2).
ACS Paragon Plus Environment
Page 10 of 26
Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Figure 4. Penetration ratio of R8 peptides and R8-NP conjugates at different concentrations. In order to further confirm the increased transmembrane efficiency of R8-NP conjugates, penetration ratio at different concentrations was calculated. The penetration ratio is defined as the ratio of the CPPs that can penetrate the membrane to the total CPPs used in the simulations. The penetration ratio of R8 peptides and R8-NP conjugates at the concentration from eleven to fifteen was given in Figure 4, with error bars showing the standard deviation. It clearly shows that the penetration ratio of R8-NP conjugates is more than that of R8 peptides at all the concentrations. This result further indicates that the translocation efficiency of CPPs can be improved by conjugating small nanoparticles with proper linkers. It may give some suggestion on designing CPPs and drugs conjugates with high transmembrane efficiency. The effect of linker length on membrane translocation of R8-NP conjugates
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 5. Lifetime of water pores (a) and the penetration ratio of R8-NP conjugates (b) at the concentration of twelve R8-NP conjugates with different length of linkers. As mentioned above, the NPs are first dragged to the water pore through the linkers and then to the cytoplasm by thermal motion. Therefore, the duration of the water pore may be relative to the length of the linkers. To shed light on the relationship between the length of the linker and the membrane translocation ability, a series of R8-NP conjugates with the linker from 0.47 to 4.23 nm (from zero to eight beads) were simulated at the concentration of twelve R8-NP conjugates. Figure 5(a) shows the statistic lifetime of water pore with different length of linkers. It shows that the lifetime of pore increases with the length of the linkers and it reaches the maximum value when the linkers are 2.4 nm, which is around half of the membrane thickness. Then it decreases as the linker length continuous increases. Figure 5(b) is the penetration ratio of R8-NP conjugates with different length of linkers. The penetration ratio was counted to 120 ns, at which almost all of the translocation of R8-NP conjugates with the linker of four beads was finished. It shows that the penetration ratio increases with increasing the length of the linkers and it reaches the maximum value when the linkers are 2.4 nm. Then it decreases with continuously increasing the linker length. When the linker is extended to 4.23 nm, the translocation ratio is even smaller than that of R8 peptides. Comparing figure 5 (a) and (b), we can see that the translocation efficiency is closely related to the lifetime of the water pore which does not only depends on the cargoes but also on the length of
ACS Paragon Plus Environment
Page 12 of 26
Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
linkers. This result implies that when CPPs are used to deliver small drugs with a covalently bonded mode, enough attention should be paid to the length of linkers.
Figure 6. Translocation process of twelve R8-NP conjugates through the asymmetric membrane with the linker of eight hydrophilic beads. (a) Time sequence of snapshots of the penetration process of R8-NP conjugates (top view, magnified side view and back view). (b) Time sequence of the center of mass separation between the R8-NP conjugates and membrane in the transmembrane direction. (c) The number of outward DMPS and inward DMPS as a function of simulation time. Based on the translocation mechanism discussed above, the lifetime of the water pore is positively relative to the length of the linkers which decide the distance between the NP and cytoplasm. In order to distinct the effect of excessive length of
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
linkers on the lifetime of water pore as well as the transmembrane efficiency, the translocation process of R8-NP conjugates with linkers of eight beads was analyzed in detail. The length of the linker with eight beads is 4.23 nm, which is comparable to the thickness of the membrane. Figure 6 (a) gives the typical snapshots of the translocation process of R8-NP conjugates through the asymmetry membrane at the concentration of twelve R8-NP conjugates. The continuous translocation is given in the animation S3. Figure 6 (b) is the COM distance between each R8-NPs conjugates and the membrane along transmembrane direction during the simulation. Figure 6 (c) is the number of outward and inward DMPS during the translocation process. We can see that at 18.4 ns the sixth R8-NP conjugate got into the water pore with the R8 peptide in front and the nanoparticle dragged behind, which is similar to that of the first R8-NP conjugate with a linker of four beads at 18.4 ns in figure 3(a). Unexpectedly, when the R8 peptide reaches the inner leaflet surface, the water pore closes with the linker buried in the membrane and the nanoparticle staying outside the membrane, as shown in the magnified cross view at 28.8 ns. The platforms from 30 ns to 70 ns in figure 6 (b) and figure 6 (c) indicate that there are no translocation of R8-NP conjugates and no transmembrane diffusion of DMPS during that time. The insertion in figure 6 (c) shows the schematic of half translocated R8-NP conjugate. When the second R8-NP conjugate migrates to the half-translocated R8-NP conjugate at around 70 ns, the water pore reopens and the second R8-NP conjugate gets to the water pore, as shown in the cross view at 80 ns. At 120 ns, only the sixth R8-NP conjugate has totally penetrated the membrane while the second and seventh R8-NP conjugates are still staying in the pore. In the six times we studied, none of the pores closed at 120 ns. To exam the subsequent translocation, the simulation time was extended to 240 ns. We find that at 240 ns the water pore is still blocked by R8-NP conjugates and the DMPS are continuously diffusing to the outer leaflet of the membrane, though four R8-NP conjugates have translocated across the membrane. The water pore formation mechanism indicates that the water pore is a result of competition between the curvature energy of membrane and the strong
ACS Paragon Plus Environment
Page 14 of 26
Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
transmembrane electric field.
13,14
In the case of R8-NP conjugate with the linker
whose length is about half of the membrane thickness, the NP has been dragged to the middle of the pore when the R8 peptide arrived at the inner leaflet surface. Contrarily, the NP remains outside membrane when the R8 peptide has arrived at the inner leaflet surface if the length of the linker is about the membrane thickness. Therefore, more curvature energy is needed to maintain the water pore with the size of the linker. That’s why the pore closes immediately after the translocation of R8 peptide with the linker buried in the membrane and the NP staying outside the membrane. But the membrane is unstable with the buried linker due to the repulsive interactions between the hydrophilic linker and the hydrophobic layer of the membrane. The pore is easy to reopen once it encounters another R8-NP conjugate. In addition, the nanoparticle with long linker will take longer time to diffuse to cytoplasm, and the pore will be easy to be blocked by the subsequent nanoparticles and does not close for even longer time. In real cells, ion transport occurs through the water pore and overlong time unclosed pore may cause serious ion leakage, which will lead to cell death.60 What is more, the asymmetry of cell membrane may be destructed if DMPS continuously diffused to the outer leaflet surface. Therefore, great attention must be paid in choosing linkers between CPPs and cargos to avoid cytotoxicity. In our simulation, similar results have been obtained when the nanoparticles are replaced by small hydrophilic molecules. Therefore, the results are mainly applicable to small hydrophilic drugs which don’t have transmembrane abilities. In this work, the standard Martini water model with long range electrostatic interactions was used. However, the polarizable Martini water model has been verified to perform the interactions between charged groups more realistically in a low-dielectric medium.61 To further investigate the effect of water model on the membrane translocation of peptides, the polarized water model with long range electrostatic interactions was used to simulate the interactions between R8 peptides (or R8-NP conjugates) and membrane. In the polarizable water model, the electrostatic interactions between the two sides of the membrane at the same concentration of
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
peptides are enhanced due to the small dielectric constant. So the concentration of R8 peptides we used in the polarized water model was six. Results in Figure S5 show that similar to those observed in standard Martini model, the translocation of the R8-NP conjugates has two stages and is slower than that of R8 peptides, which quickly penetrates to the inner leaflet of the membrane once the water pore formed. But we have to point out that the size of the water pore in the polarizable water model is a little bit larger than that in the standard water model, which makes the translocation of R8-NP conjugates quicker than before. If larger nanoparticles or cargoes were used, the rhythm of the membrane penetration such as that in the standard water model may get better. Therefore, the membrane translocation process of the two models is consistent for the present system though the electrostatic interactions are different. CONCLUSIONS The membrane translocation of R8-NP conjugates bonded with a hydrophilic linker was studied by coarse grained molecule dynamics simulations. At high concentration the R8-NP conjugates can directly translocate across the asymmetry membrane through a water pore. Different from that of R8 peptides, the membrane translocation of R8-NP conjugate with a proper linker displays a sense of rhythm. After conjugating to a hydrophilic nanoparticle, the lifetime of the water pore in the membrane is extended. As a result, the R8-NP conjugates nearby can be attracted to the pore before it is closed, which further extend the water pore’s lifetime and induce more R8-NP conjugates to translocate. Compared with R8 peptides, the translocation efficiency of R8-NP conjugates is improved and it gets the maximum value when the linker is around half of the membrane thickness. However, the R8-NP conjugates with overlong linkers will not only lower the transmembrane efficiency, but also may cause cytotoxicity due to the overlong time unclosed water pore. The results may provide some insights into the internalization of CPP-NP conjugates and facilitate the design and synthesis of new cell penetrating peptide and drug conjugates with high efficiency and low toxicity.
ACS Paragon Plus Environment
Page 16 of 26
Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Supporting Information
Results from all MD runs performed that are not presented in the main text. Animation S1-top and S1-back: Translocation of R8 peptides through asymmetry membrane. Animation S2-top and S2-back: Translocation of R8-NP conjugates through asymmetry membrane with the linker of four hydrophilic beads. Animation S3-top and S3-back: Translocation of R8-NP conjugates through asymmetry membrane with the linker of eight hydrophilic beads.
ACKNOWLEDGMENTS The work was supported by the Natural Science Foundation of Zhejiang Province (No. LQ18B040002) and Initial Scientific Research Fund of Zhejiang Sci-Tech University (No. 17062061-Y and 17062063-Y).
REFERENCES (1) Copolovici, D. M.; Langel, K.; Eriste E.; Langel, Ü. Cell-Penetrating Peptides: Design, Synthesis, and Applications. ACS Nano 2014, 8, 1972-1994. (2) Ye, J. X.; Liu, E. G.; Yu, Z. L.; Pei, X.; Chen, S. H.; Zhang, P. W.; Shin, M. C.; Gong, J. B.; He H. N.; Yang, V. C.; CPP-Assisted Intracellular Drug Delivery, What is Next? Int. J. Mol. Sci. 2016, 17, 1892. (3) Kondo, E.; Saito, K.; Tashiro, Y.; Kamide, K.; Uno, S.; Furuya, T.; Mashita, M.; Nakajima, K.; Tsumuraya,T.; Kobayashi, N. et al. Tumour Lineage-Homing Cell-Penetrating Peptides as Anticancer Molecular Delivery Systems. Nat. Commun. 2012, 3, 951. (4) Schwarze, S, R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse, Science 1999, 285, 1569-1572. (5) Li, M.; Schlesiger, S.; Knauer, S. K.; Schmuck, C. A Tailor-Made Specific Anion-Binding Motif in the Side Chain Transforms a Tetrapeptide into an Efficient Vector for Gene Delivery. Angew. Chem. Int. Ed. 2015, 54, 2941-2944. (6) Zhu, L. L.; Zhao, H. Y.; Zhou, Z. Y.; Xia, Y. H.; Wang, Z. G.; Ran, H. T.; Li P.; Ren, J. L. Peptide-Functionalized Phase-Transformation Nanoparticles for LIFU-Assisted Tumor Imaging and Therapy. Nano Lett. 2018, 18, 1831-1841. (7) Spicer, C. D.; Jumeaux, C.; Gupta, B.; Stevens, M. M. Peptide and Protein Nanoparticle Conjugates: Versatile Platforms for Biomedical Applications. Chem. Soc. Rev. 2018, 47, 3574-3620. (8) Andaloussi, S. E.; Guterstam, P.; Langel, Ü. Assessing the Delivery Efficacy and Internalization Route of Cell-Penetrating Peptides. Nat. Protoc. 2007, 2, 2043-2047. (9) Rodríguez, J.; Mosquera, J.; Couceiro, J. R.; Nitschke, J. R.; Vázquez, M. E.; Mascareñas, J. L. Anion Recognition as a Supramolecular Switch of Cell Internalization. J. Am. Chem. Soc. 2017, 139, 55-58. (10) Herce, H. D.; Garcia, A. E.; Cardoso, M. C. Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules. J. Am. Chem. Soc. 2014, 136, 17459-17467. (11)Pan, R.; Xu, W.; Ding, Y.; Lu, S.; Chen, P. Uptake Mechanism and Direct Translocation of a
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
New CPP for siRNA Delivery. Mol. Pharm. 2016, 13, 1366-1374. (12) Hirose, H.; Takeuchi, T.; Osakada, H.; Pujals, S.; Katayama, S.; Nakase, I.; Kobayashi, S.; Haraguchi, T.; Futaki, S. Transient Focal Membrane Deformation Induced by Arginine-Rich Peptides Leads to Their Direct Penetration into Cells. Mol. Ther. 2012, 20, 984-993. (13) Tang, M.; Waring, A. J.; Hong, M. Phosphate-Mediated Arginine Insertion into Lipid Membranes and Pore Formation by a Cationic Membrane Peptide from Solid-State NMR. J. Am. Chem. Soc. 2007, 129, 11438-11446. (14) Herce, H. D.; Garcia, A. E. Molecular Dynamics Simulations Suggest a Mechanism for Translocation of the HIV-1 TAT Peptide across Lipid Membranes. Proc. Natl. Acad. Sci. 2007, 104, 20805-20810. (15) Bhunia, D.; Mondal, P.; Das, G.; Saha, A.; Sengupta, P.; Jana, J.; Mohapatra, S.; Chatterjee, S.; Ghosh, S. Spatial Position Regulates Power of Tryptophan: Discovery of a Major-Groove-Specific Nuclear-Localizing, Cell-Penetrating Tetrapeptide. J. Am. Chem. Soc. 2018, 140, 1697-1714. (16) Nagel, Y. A.; Raschle, P. S. Wennemers, H. Effect of Preorganized Charge-Display on the Cell-Penetrating Properties of Cationic Peptides. Angew. Chem. Int. Ed. 2017, 56, 122-126. (17) Jobin, M. L.; Blanchet, M.; Henry, S.; Chaignepain, S.; Manigand, C.; Castano, S.; Lecomte, S.; Burlina, F.; Sagan, S.; Alves, I. D. The Role of Tryptophans on the Cellular Uptake and Membrane Interaction of Arginine-Rich Cell Penetrating Peptides. Biochimica et Biophysica Acta 2015, 1848, 593-602. (18) Du, L.; Risinger, A. L.; Mitchell, C. A.; You, J. L.; Stamps, B. W.; Pan, N.; King, J. B.; Bopassa, J. C.; Judge, S. I. V.; Yang, Z. B. et al. Unique Amalgamation of Primary and Secondary Structural Elements Transform Peptaibols into Potent Bioactive Cell-Penetrating Peptides. Proc. Natl. Acad. Sci. 2017, 114, E8957-E8966. (19) Qian, Z. Q.; Rhodes, C. A.; McCroskey, L. C.; Wen, J.; Appiah-Kubi, G.; Wang, D. J.; Guttridge, D. C.; Pei, D. H. Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization. Angew. Chem. Int. Ed. 2016, 55, 1-6. (20) Lättig-T ü nnemann, G.; Prinz, M.; Hoffmann, D.; Behlke, J.; Palm-Apergi, C.; Morano, I.; Herce, H. D.; Cardoso, M. C. Backbone Rigidity and Static Presentation of Guanidinium Groups Increases Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides, Nat. Commun. 2011, 2, 453. (21) Bagnacani, V.; Franceschi, V.; Bassi, M.; Lomazzi, M.; Donofrio, G.; Sansone, F.; Casnati, A.; Ungaro, R. Arginine Clustering on Calix[4]arene Macrocycles for Improved Cell Penetration and DNA Delivery. Nat. Commun. 2013, 4, 1721. (22) Over, B.; Matsson, P.; Tyrchan, C.; Artursson, P.; Doak, B. C.; Foley, M. A.; Hilgendorf, C.; Johnston, S. E.; IV, M. D. L.; Lewis, R. J. et al. Structural and Conformational Determinants of Macrocycle Cell Permeability, Nat. Chem. Biol. 2016, 12, 1065-1074. (23) MacEwan, S. R.; Chilkoti, A. Digital Switching of Local Arginine Density in a Genetically Encoded Self-Assembled Polypeptide Nanoparticle Controls Cellular Uptake. Nano Lett. 2012, 12, 3322-3328. (24) Macchi, S.; Signore, G.; Boccardi, C.; Rienzo, C. D.; Beltram, F.; Cardarelli, F. Spontaneous Membrane-Translocating Ppeptides: Influence of Peptide Self-Aggregation and Cargo Polarity, Sci. Rep. 2015, 5, 16914. (25) Martel, A.; Antony, L.; Gerelli, Y.; Porcar, L.; Fluitt, A.; Hoffmann, K. Q.; Kiesel, I.; Vivaudou, M.; Fragneto, G.; de Pablo, J. J. Membrane Permeation Versus Amyloidogenicity: a Multi-Technique Study of Islet Amyloid PolyPeptide Interaction with Model Membranes. J. Am. Chem. Soc. 2017, 139, 137-148. (26) Sharmin, S.; Islam, M. Z.; Karal, M. A. S; Alam, K, S. S. U.; Dohra, H.; Yamazaki, M. Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles. Biochem. 2016, 55, 4154-4165. (27) Hu, Y.; Patel, S. Thermodynamics of Cell-penetrating HIV1 TAT Peptide Insertion into PC/PS/CHOL Model Bilayers through Transmembrane Pores: the Roles of Cholesterol and Anionic Lipids. Soft Matter 2016, 12, 6716-6727. (28) Woo, S. Y.; Lee, H. Effect of Lipid Shape on Toroidal Pore Formation and Peptide Orientation in Lipid Bilayers. Phys. Chem. Chem. Phys. 2017, 19, 21340-21349. (29) Tieleman, D. P.; Leontiadou, H.; Mark, A. E.; Marrink, S. J. Simulation of Pore Formation in Lipid Bilayers by Mechanical Stress and Electric Fields. J. Am. Chem. Soc. 2003, 125, 6382-6383.
ACS Paragon Plus Environment
Page 18 of 26
Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
(30) He, X. C.; Lin, M.; Sha, B. Y.; Feng, S. S.; Shi, X. H.; Qu, Z. G.; Xu, F. Coarse-Grained Molecular Dynamics Studies of the Translocation Mechanism of Polyarginines across Asymmetric Membrane under Tension. Sci. Rep. 2015, 5, 12808. (31) Moen, E. K.; Ibey, B. L.; Beier, H. T.; Armani, A. M. Quantifying Pulsed Electric Field-Induced Membrane Nanoporation in Single Cells. Biochimica et Biophysica Acta 2016, 1858, 2795-2803. (32) El-andaloussi, S.; Järver, P.; Johansson, H. J.; Langel, Ü. Cargo-Dependent Cytotoxicity and Delivery Efficacy of Cell-Penetrating Peptides: a Comparative Study. Biochem. J. 2007, 407, 285-292. (33) Lin, J. Q.; Alexander-Katz, A. Cell Membranes Open “Doors” for Cationic Nanoparticles/Biomolecules: Insights into Uptake Kinetics. ACS Nano 2013, 7, 10799-10808. (34) Liu, X. X.; Liu, C.; Zhou, J. H.; Chen, C.; Qu, F. Q.; Rossi, J. J.; Rocchi, P.; Peng, L. Promoting siRNA Delivery via Enhanced Cellular Uptake Using an Arginine-Decorated Amphiphilic Dendrimer. Nanoscale 2015, 7, 3867-3875. (35) Liang, K.; Richardson, J. J.; Ejima, H.; Such, G. K.; Cui, J. W.; Caruso, F. Peptide-Tunable Drug Cytotoxicity via One-Step Assembled Polymer Nanoparticles. Adv. Mater. 2014, 26, 2398-2402. (36) Alhakamy, N. A.; Dhar, P.; Berkland, C. J. Charge Type, Charge Spacing, and Hydrophobicity of Arginine-Rich Cell Penetrating Peptides Dictate Gene Transfection. Mol. Pharm. 2016, 13, 1047-1057. (37) Liu, Y. Y.; Lu, Z. Z.; Mei, L.; Yu, Q. W.; Tai, X. W.; Wang, Y.; Shi, K. R.; Zhang, Z. R.; He, Q. Tandem Peptide Based on Structural Modification of Poly-Arginine for Enhancing Tumor Targeting Efficiency and Therapeutic Effect. ACS Appl. Mater. Inter. 2017, 9, 2083-2092. (38) Herce, H. D.; Schumacher, D.; Schneider, A. F. L.; Ludwig, A. K.; Mann, F. A.; Fillies, M.; Kasper, M.; Reinke, S.; Krause, E.; Leonhardt, H. et al. Cell-Permeable Nanobodies for Targeted Immunolabelling and Antigen Manipulation in Living Cells. Nature Chem.2017, 9, 762-771. (39) Wang, H.; Liu, G.; Gao, H.; Wang, Y.A pH-responsive drug delivery system with an aggregation-induced emission feature for cell imaging and intracellular drug delivery, Polym. Chem., 2015, 6, 4715-4718. (40) Lin, R.; Zhang, P. C.; Cheetham, A. G.; Walston, E.; Abadir, P.; Cui, H. G. Dual Peptide Conjugation Strategy for Improved Cellular Uptake and MitochondriaTargeting. Bioconjug. Chem. 2015, 26, 71-77. (41) Zou, X. Y.; Rajendran, M.; Magda, D.; Miller, L. W. Cytoplasmic Delivery and Selective, Multi-Component Labeling with Oligoarginine-Linked Protein tags. Bioconjug. Chem. 2015, 26, 460-465. (42) Battogtokh, G.; Cho, Y. Y.; Lee, J. Y.; Lee, H. S.; Kang, H. C. Mitochondrial-targeting anticancer agent conjugates and nanocarrier systems for cancer treatment. Frontiers in Pharmacology, 2018, 9, 922. (43) Chang, M. L.; Zhang, F.; Wei, T.; Zuo, T. T.; Guan, Y. Y.; Lin, G. M.; Shao, W. Smart Linkers in Polymer-Drug Conjugates for Tumor-Targeted Delivery. J. Drug Target. 2015, 24, 475-491. (44) Lu, J.; Jiang, F.; Lu, A. P.; Zhang, G. Linkers Having a Crucial Role in Antibody-Drug Conjugates. Int. J. Mol. Sci. 2016, 17, 561. (45) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and Challenges for the Next Generation of Antibody-Drug Conjugates. Nat. Rev. Drug Discov. 2017, 16, 315-337. (46) Doronina, S. O.; Bovee, T. D.; Meyer, D. W.; Miyamoto, J. B.; Anderson, M. E.; Morris-Tilden, C. A.; Senter, P. D. Novel Peptide Linkers for Highly Potent Antibody-Auristatin Conjugate. Bioconjugate Chem. 2008, 19, 1960-1963. (47) McCombs, J. R.; Owen, S. C. Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. AAPS J. 2015, 17, 339-351. (48) Kmiecik, S.; Gront, D.; Kolinski, M.; Wieteska, L.; Dawid, A. E.; Kolinski, A. Coarse-Grained Protein Models and Their Applications. Chem. Rev. 2016, 116, 7898-7936. (49) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111, 7812-7824. (50) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. J. The MARTINI Coarse-Grained Force Field: Extension to Proteins. J. Chem. Theory and Comput.
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
2008, 4, 819-834. (51) Uusitalo, J. J.; Ingólfsson, H. I.; Marrink, S. J.; Faustino, I. Martini Coarse-Grained Force Field: Extension to RNA. Biophys. J. 2017, 113, 246-256. (52) Dixon, J. E.; Osman, G.; Morris, G. E.; Markides, H.; Rotherham, M.; Bayoussef, Z.; El Haj, A. J.; Denning, C.; Shakesheff, K. M. Highly Efficient Delivery of Functional Cargoes by the Synergistic Effect of GAG Binding Motifs and Cell-Penetrating Peptides. Proc. Natl. Acad. Sci. 2016, 113, E291-E299. (53) Fowler, P. W.; Williamson, J. J.; Sansom, M. S. P.; Olmsted, P. D. Roles of Interleaflet Coupling and Hydrophobic Mismatch in Lipid Membrane Phase-Separation Kinetics. J. Am. Chem. Soc. 2016, 138, 11633-11642. (54) Esteban-Martín, S.; Risselada, H. J.; Salgado, J.; Marrink, S. J. Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations. J. Am. Chem. Soc. 2009, 131, 15194-15202. (55) Li, Z. L.; Ding, H. M.; Ma, Y. Q. Translocation of Polyarginines and Conjugated Nanoparticles across Asymmetric Membranes. Soft Matter 2013, 9, 1281-1286. (56) Huang, K.; Garc í a, A. E. Free Energy of Translocating an Arginine-Rich Cell-Penetrating Peptide across a Lipid Bilayer Suggests Pore Formation. Biophys. J. 2013, 104, 412-420. (57) Plimpton, S. Fast Parallel Algorithms for Short-Range Molecular Dynamics. J. comput. phys. 1995, 117, 1-19. (58) Sun, D. L.; Forsman, J.; Lund, M.; Woodward C. E., Effect of Arginine-Rich Cell Penetrating Peptides on Membrane Pore Formation and Life-Times: A Molecular Simulation Study. Phys. Chem. Chem. Phys. 2014, 16, 20785-20795. (59) Akhunzada, M. J.; Chandramouli, B.; Bhattacharjee, N.; Macchi, S.; Cardarellic, F.; Brancato, G. The Role of Tat Peptide Self-Aggregation in Membrane Pore Stabilization: Insights from a Computational Study. Phys. Chem. Chem. Phys. 2017, 19, 27603-27610. (60) Gurtovenko, A. A.; Vattulainen, I. Pore Formation Coupled to Ion Transport through Lipid Membranes as Induced by Transmembrane Ionic Charge Imbalance: Atomistic Molecular Dynamics Study. J. Am. Chem. Soc. 2005, 127, 17570-17571. (61) Yesylevskyy, S. O.; Schäfer, L. V.; Sengupta, D.; Marrink, S. J. Polarizable Water Model for the Coarse-Grained MARTINI Force Field. PLoS Comput. Biol. 2010, 6,1-17.
ACS Paragon Plus Environment
Page 20 of 26
Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Figure 1. The coarse grained structure of (a) DMPC and DMPS lipid, (b) asymmetry lipid bilayer, (c) R8 peptide, and (d) R8-NP conjugate. The nanoparticle is covalently bonded to one terminal of the backbone of R8 peptide with a hydrophilic linker (the pink beads). The types of the beads are described beside. 624x468mm (96 x 96 DPI)
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 2. Direct membrane translocation of R8 peptides and R8-NP conjugates. Top view and back view of typical snapshots of (a) the R8 peptides and (b) the R8-NP conjugates in asymmetric membrane at 0 ns and 1000 ns. Ions and water are omitted for clarity. The distance between the mass center of CPPs and membrane as a function of simulation time for (c) the R8 peptides and (d) the R8-NP conjugates along transmembrane direction at the first 240 ns. 69x49mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 22 of 26
Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Figure 3. Translocation process of twelve R8-NP conjugates through the asymmetric membrane with the linker of four hydrophilic beads. (a) Time sequence of snapshots of the translocation process of R8-NP conjugates (top view, magnified side view and back view). (b) The time evolution of center of mass distance between R8 peptides and membrane (solid lines), and nanoparticles and membrane (dotted lines) of the four R8-NP conjugates that penetrated the membrane. (c) The number of outward DMPS and inward DMPS as a function of simulation time, insert is the schematic penetration of R8-NP conjugates. 59x57mm (300 x 300 DPI)
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 4. Penetration ratio of R8 peptides and R8-NP conjugates at different concentrations. 179x145mm (150 x 150 DPI)
ACS Paragon Plus Environment
Page 24 of 26
Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Physical Chemistry
Figure 5. Lifetime of water pores (a) and the penetration ratio of R8-NP conjugates (b) at the concentration of twelve R8-NP conjugates with different length of linkers. 80x29mm (300 x 300 DPI)
ACS Paragon Plus Environment
The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 6. Translocation process of twelve R8-NP conjugates through the asymmetric membrane with the linker of eight hydrophilic beads. (a) Time sequence of snapshots of the penetration process of R8-NP conjugates (top view, magnified side view and back view). (b) Time sequence of the center of mass separation between the R8-NP conjugates and membrane in the transmembrane direction. (c) The number of outward DMPS and inward DMPS as a function of simulation time. 69x65mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 26 of 26