Reshma
Kadam
a,
Jaee
Ghawali
a,
Mario
Waespy
b,
Michael
Maas
*ac and
Kurosch
Rezwan
ac
aAdvanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany. E-mail: michael.maas@uni-bremen.de
bCenter for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of Bremen, Leobener Straße 5, NW2, 28359 Bremen, Germany
cMAPEX Centre of Materials and Processes, University of Bremen, 28359 Bremen, Germany
First published on 11th September 2020
In this study, we present Janus nanoparticles that are designed for attaching to a eukaryotic cell surface with minimal cell uptake. This contrasts the rapid uptake via various endocytosis pathways that non-passivated isotropic particles usually encounter. Firmly attaching nanoparticles onto cell surfaces for extended periods of time can be a powerful new strategy to employ functional properties of nanoparticles for non-invasive interrogation and manipulation of biological systems. To this end, we synthesized rhodamine-doped silica (SiO2) nanoparticles functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) on one hemisphere of the nanoparticle surface and high-molecular-weight long-chain poly(ethylene glycol) on the other one using the wax-Pickering emulsion technique. Nanoparticle localization was studied with NIH 3T3 rat fibroblasts in vitro. In these studies, the Janus nanoparticles adhered to the cell surface and, in contrast to isotropic control particles, only negligible uptake into the cells was observed, even after 24 h of incubation. In order to characterize the potential endocytosis pathway involved in the uptake of the Janus nanoparticles in more detail, fibroblasts and nanoparticles were incubated in the presence or absence of different endocytosis inhibitors. Our findings indicate that the Janus particles are not affected by caveolae- and receptor-mediated endocytosis and the prolonged attachment of the Janus nanoparticles is most likely the result of an incomplete macropinocytosis process. Consequently, by design, these Janus nanoparticles have the potential to firmly anchor onto cell surfaces for extended periods of time and might be utilized in various biotechnological and biomedical applications like cell surface tagging, magnetic manipulation of the cell membrane or non-invasive drug and gene delivery.
Nanoparticle uptake into cells can be mostly avoided by coating the particles with a passivation layer, which is commonly comprised of poly(ethylene glycol) (PEG).4,5 This so-called stealth coating prevents interactions with serum proteins and cell surface receptors while also reducing non-specific physico-chemical interactions between particles and cell surface molecules. On the other hand, nanoparticles can be modified with a wide variety of surface functionalizations6 to tailor the particles for specific interactions including antigen–antibody binding, receptor-mediated recognition or anchoring of lipids into the plasma membrane.1 This results in two opposing functionalization strategies: minimization of nanoparticle–cell interactions altogether via stealth coating or utilizing specific interactions that quickly result in endocytosis. The fundamental approach of the present work is to use Janus nanoparticles to incorporate both functionalization strategies on opposing hemispheres of the same nanoparticle to create particles that firmly adhere to cell surfaces without being promptly endocytosed.5,7
Janus particles, named after the Roman god with faces directed towards the past and the future, have gained popularity since their introduction by de Gennes in 19918 and have since been used in various applications like bioimaging,9 drug delivery systems,10–14 magnetotherapy15 or bacterial extraction.16,17 Janus particle preparation can be achieved by several methods including masking at interfaces,18 templating using emulsions19 and self-assembly.20 While most methods result in a relatively small number of produced Janus particles, Janus particle preparation using the wax Pickering emulsion method is a scalable method which easily yields gram quantities of approximately half-coated particles. In this approach, one hemisphere of the particle is masked at the surface of solidified wax emulsion droplets and hence the exposed particle hemisphere can be modified with a separate functionality.21–23
In a seminal paper from 20137 and its follow-up from 2015,24 Gao and Yu explored the endocytosis and fate of micron-sized Janus particles which were coated with anti-CD3 antibodies and a passivation layer of bovine serum albumin (BSA) on respective sides. In this first detailed study on the uptake of Janus particles, the authors were able to show a two-step endocytosis pathway in which the antibody-coated side was progressively engulfed via receptor-mediated endocytosis. Afterwards the BSA-coated side was covered with a further extension of the cell membrane in an actin-mediated process. Full endocytosis of these Janus microparticles by Jurkat T cells was reported to take 1–5 min, which was significantly longer than endocytosis of isotropic particles. In a similar approach, Sanchez et al. studied the effect of PEG functionalization on one hemisphere of Janus microparticles on their uptake by macrophages. The passivation with PEG instead of BSA resulted in strongly reduced internalization of about 25% of the incubated particles measured after 30 min. With anti-CD3 antibodies on the binding hemisphere, they report that also about 25% of the particles remain bound to the cell surface after an incubation time of 30 min.5 Note that the reported experimental studies have been carried out with micron-sized particles whose anisotropic features are easily visualized with fluorescence microscopy. Compared to microparticles, nanoparticles interact significantly different with biological systems due to their much higher surface area to volume ratio and dimensions that are much closer to proteins which might lead to drastically different uptake behavior.25 Additionally, a fairly large body of computational studies about Janus particle interactions with phospholipid membranes provide theoretical background to some aspects of these observations which are summarized in a recent review.26
Here, we will build on the findings for microscale Janus particles and introduce a nanoscale Janus particle design, which features membrane anchoring phospholipids on one hemisphere and a PEG stealth coating on the other. By creating such dual-functionalized particles, we aim to anchor the nanoparticles onto eukaryotic cell membranes and at the same time to inhibit the endocytosis process that leads to uptake of these particles into cells via the passivation layer on the other half of the particle surface. To this end, we synthesized dye-doped silica nanoparticles (50 nm diameter) functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) on one side and PEG on the other side of the particle surface. To facilitate insertion of the phospholipid DSPE into the cell membrane, it was connected to the particle surface via a flexible PEG-linker. The particles were first characterized in terms of their material properties and colloidal behavior. In in vitro cell culture studies, the Janus nanoparticles were analyzed jointly with their isotropic (non-Janus) pendants as controls in a mouse fibroblasts cell culture model using NIH 3T3 cells regarding their association to the plasma membrane and their endocytosis properties. Cellular location and distribution patterns of the particles were determined by confocal laser scanning microscopy (CLSM) coupled with Z-stack imaging and transmission electron microscopy (TEM). Furthermore, we incubated the nanoparticles together with NIH 3T3 cells in the presence or absence of various endocytose inhibitors to reveal associated internalization pathways.
In vitro cell culture experiments were carried out using the mouse fibroblast cell line NIH 3T3 (passage number 8–25, product no. 400101), which was purchased from CLS Cell Lines Services GmbH (Eppelheim, Germany) and cultured in Dulbecco's Vogt modified Eagle's minimum essential medium (DMEM; high glucose, lot no. 1206393) supplemented with fetal calf serum (FCS, product no. 010M3395) and antibiotic–antimycotic solution (AB/AM, product no. 1209917) were purchased from Invitrogen (Germany). Trypsin-ethylenediaminetetraacetic acid (0.25% trypsin, 0.02% EDTA, product no. SLBG4376), Triton X-100 (product no. MKBL5839 V), phosphate buffered saline (PBS, product no. SLBF5741 V), paraformaldehyde (PFA, product no. 53260), osmium tetroxide solution (OsO4, 4% in water, product no. 75632), glutaraldehyde solution (Grade, 1.25% in water, product no. G5882), 4′,6-diamidino-2-phenylindole (DAPI, 0.5 μg ml−1, product no. 1242642), Alexa fluor 488 phalloidin (AF488, 2 U ml−1, product no. 1151587), wortmannin (lot no. 023M4072), nystatin (lot no. 020M13491), sucrose (lot no. 1076511000), agar (lot no. A1296) and an epoxy embedding medium kit (Epon 812 substitute, product no. 45359) were purchased from Sigma Aldrich (Germany). Cell toxicity was measured using water-soluble tetrazolium salt assay (WST-1, Roche Diagnostics GmbH, Germany, product no. 14310100) and lactate dehydrogenase assay (LDH), Pierce assay (Thermo Scientific, Germany, lot no. OL17881450).
The density of the grafted NH2 groups on NH2-functionalized RBITC-doped SiO2 was then quantified spectrophotometrically using the reagent ninhydrin by adapting the procedure described by Soto-Cantu et al.29 The nanoparticles were dispersed in absolute ethanol at a concentration of 10 mg ml−1 using ultrasonication for 10 min. 200 μl of 0.35% (w/v) solution of ninhydrin in ethanol was added to the particle solution followed by ultrasonication for 5 min. This sonicated ninhydrin–nanoparticle mixture was incubated in a heating bath at 65 °C for 5 min, which was further left to cool for 5 min. The Eppendorf tubes were centrifuged to remove any unreacted ninhydrin molecules at 13000 rpm for 20 min. The absorbance of 1 ml of the supernatant was then measured using a Multiscan Go spectrometer at 588 nm. The amount of unbound ninhydrin was used to calculate the amount of bound ninhydrin, from which the amount of NH2 groups per nm2 of the surface area of the nanoparticles was deducted. Increasing concentrations of a standard solution of hexylamine (0.12 mM to 0.87 mM) was used to prepare a calibration curve. All particle types were measured in triplicates and the amount of amine functional groups per nm2 were expressed along with standard deviations.
With the same approach, further nanoparticles have been prepared with and without Janus functionalization for control experiments: isotropic unfunctionalized particles, fully APTES-coated particles, fully PEG-coated particles, fully DSPE-coated particles and anisotropic APTES/PEG Janus nanoparticles. Isotropic unfunctionalized particles were utilized as prepared. The preparation of the fully APTES-coated particles is mentioned above. For the preparation of fully PEG-coated particles, 5 ml of 10 mg ml−1 RBITC-doped SiO2 nanoparticles in ethanol was incubated with 40 μl of mPEG silane incubated for 8 h under stirring at room temperature.32 The silanized particles were washed several times using absolute ethanol and ddH2O using alternative centrifugation and redispersion cycles. In case of the fully DSPE-coated particles, 5 ml of 10 mg ml−1 fully APTES-coated particles in ethanol were incubated with 40 μl of DSPE-PEG-COOH using the EDC-NHS coupling procedure.
PEG functionalization of the nanoparticles was assessed by measuring the adsorption of FITC-BSA. 10 mg nanoparticles were dispersed in 1 mM PBS and incubated with 2 μmol of FITC-BSA per gram of nanoparticles for 1 h at room temperature. This mixture was then washed thrice with ethanol–water solution to remove unbound FITC-BSA. The residual and adsorbed FITC-BSA concentrations were then quantified using a fluorescence plate reader (Chameleon V, Hidex, Germany). By measuring the fluorescence signal at λex: 495 nm, λem: 525 nm of known increasing concentrations of FITC-BSA (1 nmol to 20 nmol FITC ml−1 of PBS), a calibration curve was prepared, which was used to calculate unknown FITC-BSA concentrations of the samples.
Intra- and extracellular quantities of lactate dehydrogenase (LDH) were quantified in the absence and presence of the nanoparticle dispersions using the Pierce assay.33,36 For the quantification of extracellular LDH released by damaged cells due to the loss of membrane integrity, the media was collected after incubation of the cells with the nanoparticles and centrifuged at 10000g for 3 min to remove any dispersed nanoparticles. The quantification of released LDH was performed following supplier instructions using 50 μL of cell culture medium. To obtain the intracellular LDH concentration, cells were detached from cell culture plates by trypsin-EDTA treatment and centrifuged at 600g for 10 min to harvest the cells. Afterwards, cell lysis was performed using lysis solution (1% v/v Triton X-100 in 0.9% w/v NaCl in water). The suspension was mixed by pipetting several times to obtain a clear solution and 50 μl of the cell lysate was used for the LDH assay. After incubation for 30 min in the dark, the absorbance of the samples after addition of 50 μl of the assay solution was analyzed at 490 nm along with a reference measurement at 680 nm using a Multiscan GO spectrophotometer. Cells incubated in DMEM + FCS (10%) and AB/AM (1%) were used as minimum LDH activity control (100% cell viability) and cells incubated with 2% v/v Triton X-100 in 1 mM PBS buffer were used as maximum LDH activity control (0% cell viability). The amount of LDH was normalized to the total LDH concentration (extracellular LDH + intracellular LDH).
After the cooling and washing steps, the solvent-exposed particle hemispheres on the solidified wax droplets are functionalized with PEG-silane. Afterwards, the wax is dissolved and the particles are thereby released from the wax spheres. The originally masked NH2 groups are then functionalized with COOH-PEG-DSPE via EDC-NHS chemistry. Additionally, further isotropic and Janus nanoparticles were prepared as listed in Table 1 as controls for the in vitro cell culture studies.
Liquid medium | Unfunctionalized | APTES | APTES/PEG | PEG | DSPE | DSPE/PEG | |
---|---|---|---|---|---|---|---|
Particle size (nm) | 4% ethanol/water | 50.5 ± 6.2 | 54.1 ± 6.4 | 65.5 ± 7.8 | 60.4 ± 10.8 | 85.6 ± 16.4 | 86.2 ± 10.4 |
DMEM + FCS + AB/AM | 52.4 ± 7.3 | 62.4 ± 4.3 | 67.2 ± 2.3 | 58.7 ± 2.6 | 86.9 ± 7.8 | 89.7 ± 3.2 | |
PDI | 4% ethanol/water | 0.13 ± 0.03 | 0.15 ± 0.04 | 0.18 ± 0.01 | 0.14 ± 0.09 | 0.25 ± 0.05 | 0.21 ± 0.03 |
DMEM + FCS + AB/AM | 0.21 ± 0.12 | 0.22 ± 0.08 | 0.17 ± 0.06 | 0.21 ± 0.05 | 0.24 ± 0.13 | 0.24 ± 0.07 | |
Zeta potential (mV) | 4% ethanol/water | −31.4 ± 4.4 | 7.4 ± 8.7 | −26.3 ± 1.6 | −32.5 ± 7.4 | −35.2 ± 6.7 | −40.4 ± 6.4 |
DMEM + FCS + AB/AM | −16.7 ± 2.6 | −8.3 ± 2.1 | −7.6 ± 4.2 | −30.4 ± 6.2 | −20.4 ± 5.6 | −24.4 ± 3.7 | |
NH2 groups/surface area (nm2) | Water | 1.8 ± 0.3 | 20.7 ± 6.8 | 15.3 ± 7.4 | 4.3 ± 1.8 | 4.5 ± 3.4 | 2.5 ± 1.6 |
The prepared particles were characterized regarding their size and zeta potential by dynamic light scattering (DLS) before they were used for cell experiments. In order to assess the behavior of the particle dispersion during the cell culture experiments, the particles were analyzed both as a dispersion in a 4 vol% ethanol/water mixture and dispersed in the cell culture medium (DMEM + FCS (10%) + AB/AM (1%)) which was later used in the cell culture studies. DLS analysis of the unfunctionalized RBITC SiO2 nanoparticles dispersed in the ethanol/water mixture revealed a size of 50.5 ± 6 nm (Table 1). A similar hydrodynamic diameter of 52.4 ± 7 nm (Table 1) was recorded when these nanoparticles were dispersed in the cell culture medium, showing that colloidal stability was maintained under these conditions. However, APTES-functionalization of the RBITC SiO2 nanoparticles resulted in a moderate increase of the particle sizes in cell culture medium (62.4 ± 4 nm) indicating slightly decreased colloidal stability, possibly as a result of protein adsorption (mainly BSA as the main constituent of FCS) on the positively charged nanoparticle surface.37 Isotropic PEG functionalization resulted in nanoparticles with a hydrodynamic diameter of 60.4 ± 10.8 nm measured in ethanol/water (Table 1). In addition, DLS analysis of PEG-functionalized nanoparticles in cell culture medium lead to a similar hydrodynamic diameter of 58.7 ± 2.6 nm, most likely due to the suppressed protein adsorption of the PEGylated particle surface.33 The slight increase in hydrodynamic diameter of PEGylated nanoparticles relative to that of unfunctionalized nanoparticles can in this case be attributed to the long-chain PEG groups functionalized on the SiO2 nanoparticles. The APTES/PEG Janus nanoparticles show somewhat decreased colloidal stability with higher hydrodynamic radius as a result of the anisotropic functionalization. Because of their increased hydrophobicity compared to the hydrophilic unfunctionalized and APTES-functionalized particles, fully DSPE-coated particles show again higher particle sizes and wider size distribution indicated by an increased polydispersity index (PDI) of 0.25 possibly due to agglomeration of the hydrophobic nanoparticles in the aqueous medium. Finally, the DSPE/PEG Janus nanoparticles displayed a hydrodynamic diameter of 86.2 ± 10.4 nm and 89.7 ± 3 nm in ethanol/water and cell culture medium, respectively (Table 1). Along this line, we most likely see some aggregation of DSPE/PEG Janus nanoparticles in the form of a mixture of dimeric agglomerates and individually dispersed particles possibly as a consequence of both the increased hydrophobicity of the DSPE and the Janus character of the particles. We expect that dimeric particles with the DSPE hemispheres pointing towards each other will behave similar to fully PEG-coated particles in subsequent experiments. Note that TEM analysis of the agglomeration state of the Janus particles is not suitable, since the drying process during sample preparation causes agglomeration, as well. The size of the DSPE/PEG nanoparticles seems to be unaffected by the presence of the otherwise surface-active molecules in the cell culture medium.
These findings are further substantiated by zeta potential measurements of all nanoparticle types in ethanol/water and cell culture medium, respectively (Table 1). Of special note is the positive surface charge of APTES-coated nanoparticles in ethanol/water which reverts to a negative charge in cell culture medium as a result of protein adsorption.32,37 Correspondingly, a decrease in zeta potential was observed for the APTES/PEG Janus nanoparticles resuspended in cell culture medium. In all other cases, regardless of the dispersion medium, the nanoparticles remained negatively charged.
Quantification of primary amino groups on the as-prepared nanoparticles was performed using the ninhydrin assay. Here, 20.7 ± 6.8 NH2 groups per nm2 of the surface of NH2-functionalized SiO2 nanoparticles were determined and Janus functionalization of the APTES/PEG nanoparticles reduced the number of quantifiable NH2 groups to 15.3 ± 7.4 amino groups per nm2 of the particle surface.
The success of the PEG-functionalization was further assessed by incubating the particles with FITC-BSA and analyzing the ratio of FITC-BSA adsorbed to the surface of nanoparticles to dissolved FITC-BSA in the supernatant (Fig. S2†). After 1 h of incubation, unfunctionalized and APTES-functionalized nanoparticles showed high amounts of FITC-BSA adsorption (Fig. S2A,† black bars), whereas negligible amounts of BSA were detected in the respective supernatant (Fig. S2B,† grey bars). On the other hand, APTES/PEG functionalized nanoparticles showed lower amounts of adsorbed FITC-BSA in comparison to the unfunctionalized and the positively charged APTES functionalized nanoparticles. As control, adsorption of FITC-BSA to PEG functionalized RBITC SiO2 nanoparticles was measured. Low amounts of FITC-BSA on the nanoparticles were detected confirming the success of PEG functionalization.
For FITC-BSA adsorption studies on both DSPE- and DSPE/PEG functionalized particles, negligible amounts of FITC-BSA adsorption was observed on both particle types. The amounts of BSA adsorption are comparable to those obtained with fully PEG functionalized particles, showing that the DSPE-coating also prevents BSA adsorption.
Lastly, direct visualization of the immobilized organic molecules on the surface of the nanoparticles is extremely difficult using TEM due to their poor contrast. In a previous study, we grafted proteins bearing ultra-small gold or iron oxide nanoparticles on Janus particles to provide contrast in TEM, which more directly demonstrated the success of our approach.23 While the wax Pickering emulsion technique achieves the preparation of bulk quantities of Janus particles, we also observed that, with nanoparticles, the method does not provide a well-defined Janus balance.16,23
When incubated with DSPE/PEG Janus nanoparticles, the nanoparticles remained constrained to the outer regions of the cells at all studied time points (Fig. 3A–F). Conversely, when cells were incubated with fully DSPE-functionalized nanoparticles, at 1 h (Fig. 3A and B), nanoparticle accumulation was observed on the cell surfaces. After 6 h of incubation (Fig. 3C and D), the nanoparticles were taken up into cellular microvesicles which was even more pronounced after 24 h of incubation (Fig. 3E and F). Most strikingly, a clear distinction between DSPE/PEG- and DSPE-functionalized nanoparticles was observed after 24 h of incubation. The majority of DSPE-functionalized nanoparticles were localized inside the cells and, in contrast, when cells were incubated with DSPE/PEG Janus particles, almost no internalization was observed (see Fig. S6† for further images with these particles). Additionally, the DSPE/PEG Janus nanoparticles seem to be mainly located at extracellular ruffled protrusions of the cell membrane which could possibly be associated to filipodia of the plasma membrane. Fig. S7 and S8† illustrates the internalization of all other types (see Table 1) of synthesized nanoparticles which is clearly visible already after 1 h of incubation, as expected.
As expected, without nanoparticles and with fully PEG-coated particles, no particles are visible in any experiments (Fig. 4, first two rows). As discussed above, unfunctionalized and APTES-coated nanoparticles were strongly internalized in the absence of endocytosis inhibitors (Fig. 4, third row, see also Fig. S5 and S8†). In direct comparison, DSPE- and DSPE/PEG-functionalized nanoparticles seem to be taken up to a lesser degree than APTES-coated and unfunctionalized nanoparticles (Fig. 4, fourth and fifth row). However, on the basis of the fluorescent microscopy images it is difficult to make a distinction between surface-attached and internalized nanoparticles. We assume that all visible extracellular nanoparticles are somehow associated to the cell surface since these still remained after the washing steps. Temperature-dependent suppression of endocytosis by incubating NIH 3T3 cells at 4 °C (Fig. 4, second column) caused a strong decrease or slowdown in the uptake of the unfunctionalized and APTES-functionalized particles. This mitigation of uptake was also detected for DSPE- and DSPE/PEG-functionalized nanoparticles after 1 h of incubation (Fig. S9†), but is no longer visible after 6 h and 24 h (Fig. 4 and Fig. S10†). Incubation of NIH 3T3 cells in hypertonic media containing 0.45 M sucrose should overload cell surface receptors involved in receptor-mediated endocytosis (Fig. 4, third column).38 This treatment also resulted in lower uptake rates of unfunctionalized and APTES-functionalized nanoparticles relative to those observed for untreated samples incubated under standard culturing conditions (Fig. 4, first column). However, cellular uptake of DSPE- and DSPE/PEG-functionalized nanoparticles remained unaffected by the high-sucrose medium treatment. The same trend can be observed when the endocytosis inhibitor nystatin is added to the cell culture medium (Fig. 4, fourth column). Nystatin is known to inhibit caveola-dependent endocytosis and seems to be exclusively effective to prevent cellular uptake of the hydrophilic unfunctionalized and APTES-functionalized nanoparticles. Along this line, in the presence of wortmannin (Fig. 4, fifth column), a well-characterized macropinocytosis inhibitor, no intracellular accumulation was observed for any nanoparticle type.
While a straightforward interpretation of these results is difficult since several endocytosis pathways might be active for one particle type, they indicate that both caveolae-dependent endocytosis and macropinocytosis are involved in the uptake of isotropic APTES-coated and unfunctionalized nanoparticles. The interaction of NIH 3T3 cells with isotropic DSPE- and DSPE/PEG-functionalized Janus nanoparticles is not strongly affected by the presence of nystatin or the high sucrose concentration. Conversely, suppression of macropinocytosis via wortmannin inhibited the association of both DSPE- and DSPE/PEG-functionalized nanoparticles. These results indicate that macropinocytosis seems to be the main endocytosis pathway for the uptake of the fully DSPE-coated nanoparticles. Although the TEM analysis (Fig. 3) suggests that the DSPE/PEG-functionalized Janus nanoparticles are mainly attached to the cell surface and are only barely taken up to some extent, some effects of macropinocytosis seem to be involved in firmly attaching the nanoparticles to the cell surface. This is to some degree substantiated by the observation of the roughened cell surface in the presence of the Janus nanoparticles in the TEM micrographs (Fig. 3A–F). These results could be an evidence for an interrupted macropinocytosis process that accumulates the nanoparticles at the cell surface without forming closed macropinosomes and subsequent transfer into the cytosol.
Finally, anisotropic particles that are designed to stay permanently attached to membrane surfaces might prove eminently useful in biomedical applications. Particularly magnetized Janus nanoparticles could be able to move or otherwise manipulate individual cells non-invasively by serving as a magnetic handle at the cell membrane leading to novel tools for the field of magnetic tissue engineering.39 Furthermore, nature has already brought forth anisotropic nanoparticles that are able to inject their DNA or RNA into cells after latching onto cell surfaces. Accordingly, myovirus bacteriophages and similar viruses could act as templates for even more sophisticated anisotropic nanoparticle drug or gene delivery systems in the future.
Footnote |
† Electronic supplementary information (ESI) available: Supplementary figures. See DOI: 10.1039/d0nr04061d |
This journal is © The Royal Society of Chemistry 2020 |