Calcium condensation of Tat/pDNA complexes into nontoxic delivery nanoparticles
To reduce the toxicity and at same time to improve the gene delivery efficiency of Tat/pDNA complex, calcium chloride was used as a compact agent to condense the particles of Tat/pGL3 complex. Gel retardation assay shown that Ca2+ addition (final conc. 113 mM) efficiently condensed Tat/pGL3 or Tat/pGL3-DiYO-1 complexes into nanoparticles even when the N/P ratio reached to 1 (Fig. 1A, B). This is a significant improvement from our previous observation that Tat-facilitated pDNA compact formation only occurred when N/P ratios more than 5 [6]. Condensation ability of Ca2+ addition was consistent when the N/P ratio of Tat/pDNA complexes increased from 1 to 20. Condensing stability of Tat/pDNA-Ca2+ nanoparticles was determined by DNase I protection assay. Total resistance to DNase I action was observed for Tat/pGL3-Ca2+ nanoparticles induced at N/P ratios not less than 1 (Fig. 1C). DLS (dynamic light scattering) measurements showed that formulated Tat/pGL3-Ca2+ nanoparticles presented weak positive ζ potential (average 3.8 mV), and possessed small particle size (252–512 nm) at N/P ratio of 10 (Fig. 1D. Additional file 1: Table S1. Additional file 2: Fig. S1). These results are similar to previous findings [11, 30], highlighting the effect of Ca2+ addition to Tat/pDNA complexes induced substantial decrease in the particle size and competitively inhibit the amine/phosphate interaction. This synergic effect might be attributed to the “soft” condensation ability of Ca2+ to Tat/pDNA complexes, because defined concentration range of calcium interactions with both amines (polycations) and phosphates (DNA) can control particle size [11, 31, 32]. The uneven size-distribution of Tat/pGL3-Ca2+ nanoparticles is compatible with the results from AFM and TEM checking images, and irregular granules with asymmetric morphology were frequently observed (Fig. 1E–H). High positive ζ potential and small diameter of Tat/pGL3-Ca2+ nanoparticles were reached with enhanced N/P ratios (Additional file 1: Table S1). Without Ca2+ addition, we only observed big, loose complexes between Tat peptide and pDNA [6], whereas small, compact Tat/pDNA nanoparticles were formed after Ca2+ addition (Fig. 1E–H. Additional file 1: Table S1).
Compared to previous formulation [6, 7], adding Ca2+ in this work led to high-efficient delivery of Tat/pDNA complexes without apparent toxicity (Figs. 2, 7E, Additional file 2: Fig. S2, S13). Less apoptosis-induced in OC cells and negligible body weight or visceral changes in OC-bearing mice were observed after treatment with Tat/pDNA-Ca2+ nanoparticles. Noticeably, improved cell survival was observed after adding Ca2+ into Tat/pDNA complex (Additional file 2: Fig. S2C-D), and only high-concentration (20ug/mL, NP = 10), long-term (14 h) exposure of Tat/pGL3-Ca2+ nanoparticles to cells can trigger necrotic apoptosis (Additional file 2: Fig. S2A-B). It was reported that Ca2+ addition (> 30 mM) can reduce aggregation and yield of more monodisperse lipoplexes [33]. We observed agglomeration of Tat/pDNA complexes without CaCl2, whereas Tat/pDNA-Ca2+ nanoparticles with optimal CaCl2 concentration (113 mM) were relatively stable in mouse serum and serum-contained culture media (Additional file 2: Fig. S3). This stability was also demonstrated by the heparin displacement assay, because exposing the Tat/(SYBR Green)-pDNA-Ca2+ nanoparticles to the highly negatively charged heparin yielded an increase in the fluorescence signal with increase in heparin concentration (Additional file 2: Fig. S4). Stable body weight of mice and no visceral toxicity in OC-bearing mice (Additional file 2: Fig. S13, Fig. 7E), indicate that the formulated Tat/pDNA-Ca2+ nanoparticles are safe. These results demonstrated that Tat/pDNA complexes were tightly condensed into small, nontoxic delivery nanoparticles by Ca2+ addition.
Cell-cycle-dependent macropinocytic uptake of Tat/pDNA-Ca2+ nanoparticles
Macropinocytosis was reported as a dominant uptake route for Tat-based gene delivery vectors [6, 7, 16, 34,35,36]. Here, we found that Tat/pGL3-Ca2+ nanoparticles mainly utilized macropinocytosis for uptake in OC cells. Using various endocytic markers as indicated in the literatures, confocal imaging showed that Tat/pGL3-Ca2+ nanoparticles were not co-localized with the clathrin-mediated endocytosis (CME) marker Tfn-AF647 and lipid raft marker CTxB, but co-localized with the macropinocytosis marker Dextran (Fig. 2A, Additional file 2: Fig. S5A, S6). SKOV3 cells pretreated with CME inhibitor CPZ, or caveolae-mediated endocytosis (CvME) inhibitor filipin, or cholesterol-depleting reagent MBCD did not significantly inhibit the uptake of Tat/pGL3-Ca2+ nanoparticles. In contrast, when cells were pretreated either with the macropinocytosis inhibitor EIPA or with CyD, the uptake of nanoparticles was significantly inhibited (Additional file 2: Fig. S5B). These results were similar to our previous observations [6, 7, 27]. The phenomenon that Tat/pDNA-Ca2+ nanoparticles preferred macropinocytic uptake in OC cells can be explained by: (1) since macropinocytic capacity is determined by the properties of cells [14, 15], intensified macropinocytosis of Tat/pDNA complex was induced in OC cells with certain mutations [27]. (2) Positive-charged, botryoid-shaped nanoparticles enter tumor cells via macropinocytosis more efficiently than negative-charged, shape-defined nanoparticles [17, 37]. Our Tat/pDNA-Ca2+ nanoparticles with irregular and asymmetric morphology (Fig. 1E, H) largely manipulate macropinocytosis to favor their intracellular delivery; (3) Cationic CPPs binding with anionic glycosaminoglycans (GAGs) on cell surface always stimulate Rac activation, which induces actin polymerization, lamellipodia formation and subsequent macropinocytosis-initiation [20, 38, 39]. Excess Tat peptide within Tat/pDNA-Ca2+ nanoparticle (NP > 10) are prone to stimulate macropinocytosis boosted in OC cells (Fig. 2, 4); (4) Cancer cells undergoing blebbishield emergency program exhibit robust macropinocytosis [40], the pro-apoptotic status of OC cells stimulated by Tat/pDNA-Ca2+ nanoparticles (Additional file 2: Fig. S2) may further promote the macropinocytosis in these cells. In addition, we also observed macropinocytic difference between Skov3 and Cos7 cells (Fig. 2K, L, Fig. 4A–D), suggesting the cell-specific uptake of Tat/pDNA-Ca2+ nanoparticles [4, 32], which is of importance for design treatment of different cell types.
Different from the prevailing claims that pinocytosis is shut down during mitosis [41,42,43], we found here that macropinocytosis was decreased but not completely arrested in cell-cycle M-phase. Confocal microscopic examining the co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in unperturbed cells showed that they were still internalized via macropinocytosis in different sub-phases of cell-cycle (Fig. 2A–H). Macropinocytic uptake of Tat/pGL3-Ca2+ nanoparticles appeared frequently shutdown in cells at I-phase (interphase) (Fig. 2B–D). To clarify these variations, we synchronized cells at distinct sub-phases of cell-cycle by chemotherapeutics pretreatments (Fig. 2E). Confocal microscopic examining the co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in arrested cell-cycle phases showed that their macropinocytosis were markedly increased in GCB(gemcitabine)-induced S-phase and persisted in PTX(paclitaxel)-induced M-phase (Fig. 2F–H). Determining these variations by fluorescence quantification assay also confirmed the enhanced co-uptake of Tat/pGL3-Ca2+ nanoparticles with Dextran in cells at S- and M-phases (Fig. 2I, J). Addition of macropinocytosis inhibitor EIPA (5-(N-ethyl-N-isopropyi)-amiloride) totally reversed this enhancement (Additional file 2: Fig. S6A, B). Further checking the impact of Tat/pDNA-Ca2+ nanoparticles on cell-cycle distribution found that sub-phases of asynchronous or synchronized cells were not significantly altered after incubating with Tat/pGL3-Ca2+ nanoparticles (Additional file 2: Fig. S7). Additionally, enhanced transgene expression of Tat/pGL3-Ca2+ nanoparticles in S- or M-phase cells and transduction-inhibition by EIPA co-incubation were observed (Fig. 2K, L, Additional file 2: Fig. S6C). These results suggest that macropinocytosis and expression of Tat/pDNA-Ca2+ nanoparticles were persisted in cell-cycle M-phase and markedly increased in cell-cycle S-phase.
Previous studies showed that several cellular uptake pathways (caveolar and clathrin-mediated endocytosis) do take place during normal mitosis when checking asynchronous cells under advanced microscopy (e.g. Electron microscopy) [44,45,46]. Considering only 0.5–2% of mammalian cells undergoing mitosis and residual endocytosis are dedicated to the turnover of plasma membrane and specific receptors during the successive process (prophase, metaphase, anaphase, telophase and cytokinesis) [42, 47], re-activated macropinocytosis of Tat/pGL3-Ca2+ nanoparticles in partial cell population at M-phase are logical (Fig. 2B–H). Additionally, upregulated expression of macropinocytosis driver Arf6 at M-phase (Fig. 3B) also suggests that reactivated macropinocytosis is liable to support plasma membrane remodeling when cells undergoing division. Notably, the finding here is that macropinocytosis of Tat/pGL3-Ca2+ nanoparticles was highly upregulated in GCB-induced S-phase cells (Fig. 2B–H). To our knowledge, this is the first observation that macropinocytosis peaked up at specific phases of cell-cycle. In vivo data with enhanced delivery and killing efficiency of Tat/TF-Ca2+ nanoparticles in GCB-administrated group also confirmed this finding (Figs. 7C, D, 8A, B). Little is known about the factors accounting for this contrast phenomenon. As intact nuclear membrane and undersized nuclear pore complex (10–26 nm) are impossible to assist the nuclear uptake of Tat/pDNA-Ca2+ nanoparticles when transfection was carried out at this phase, possibly factors may be from certain stages of macropinocytosis. High macropinocytosis of Tat/pDNA-Ca2+ nanoparticles but low Arf6 expression at S-phase appears paradoxical (Fig. 2F–H, Fig. 3B). Since Arf6 can regulate macropinocytosis and also act on the terminal stage of cytokinesis [48, 49], highly expression and accumulation of Arf6 at M-phase is beneficial for its localizing at cleavage furrow and midbody so as to finish the cytokinesis process. Although activated Arf6 (GTP binding) is required for cell surface recycling of short-lived macropinosome, inactivation of Arf6 (GTP hydrolysis) is essential to intracellular macropinosome trafficking [31, 50], therefore, low expression of Arf6 at S-phase contributes to active macropinocytosis is logical. In addition, Tat-based nanoparticle bound with cell surface GAGs is an essential prerequisite for their uptake [38]. Because the structural and quantitative differences of GAGs (especially on the cell surface) during cell-cycle are still debated [33, 51], it’s hard to relate macropinocytosis kinetics to the expression profile of GAGs in the cells, yet.
GRP75 driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles
Seeing that cell-cycle and endocytosis-transition are synchronously regulated by moonlighting chaperones [24, 52, 53], we next determined whether mitochondrial moonlighting chaperone GRP75 acting on the cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Cyclebase-based predication showed that high-expression of GRP75 frequently appeared at S-phase and G2(gap phase 2)/M boundary. High-expression of GRP75-bound mitotic kinase MPS1 was mainly distributed from S- to M-phase. However, high-expression of GRP75-bound, MPS1-recruited mitochondrial gatekeeper VDAC1 was mainly presented at S-phase. Remarkably, high-expression of macropinocytosis driver Arf6 was scarcely distributed at S-phase, G2/M boundary and early G1-phase (gap phase 1) (Fig. 3A). Western blot analysis of cell fractions showed that centrosome-associated GRP75 and MPS1 were highly enriched at S-, G2-, and M-phases than that at G1-phase. In contrast, centrosome-associated VDAC1 and Arf6 were sharply reduced at these phases (Fig. 3B). Unexpectedly, whole lysate-derived and cytoplasmic VDAC1, Arf6 were reduced at S-phases, and no significant expression-difference of GRP75 and MPS1 was detected. These results suggest that GRP75 and MPS1 co-enriched at centrosome from cell-cycle S- to M-phase.
GRP75 was shown being associated with duplicated centrosomes, phosphorylated by Mps1 on Thr62 and Ser65, and feedback super-activated Mps1 in HeLa-1 and U2OS cells [54, 55]. To determine whether it could moonlight as a regulator on cell-cycle and macropinocytosis, we created lentivirus stable-transfected Cos7 and Skov3 cells with GRP75-knock-down (KD) and –over-expression (OE) of its phosphorylation mutants (Fig. 3C, D, Additional file 2: Fig. S8). Flow cytometry analysis showed that GRP75-KD or phosphorylation-inactivation (T62A/S65A) significantly induced cell-cycle accumulated at G1-phase. In contrast, GRP75-OE or phosphorylation-activation (T62D/S65D) promoted cell-cycle S-phase enriched and M-phase arrested (Fig. 3E). Confocal checking the uptake of Tat/pGL3-Ca2+ nanoparticles showed that their macropinocytosis were markedly reduced in GRP75-KD or phosphorylation-inactivation (T62A/S65A) cells, but significantly enhanced in GRP75-OE or phosphorylation-activation (T62D/S65D) cells (Fig. 4A, B). Such distinct uptake of Tat/pGL3-Ca2+ nanoparticles in GRP75-interrupted cells was also observed in fluorescence quantification assays (Fig. 4C), and correspondingly resulted in the variation of transgene expression (Fig. 4D). Additionally, distinct macropinocytosis of Tat/pGL3-Ca2+ nanoparticles were observed in EGFP-positive, GRP75-transiently-transfection cells (Fig. 4E, F). These results suggest that cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticle was driven by high-expression or phosphorylation of GRP75.
Although further work is needed to deeply dissect why macropinocytosis was highly upregulated by GRP75 and its phosphorylation (Fig. 4), the potential possibilities can be: (1) GRP75-OE increase the expression of S-phase transcription factor E2F-1 and cyclin-dependent kinase inhibitor p21 [47]. P21-activated kinase 1 (Pak-1) can promote CPPs uptake and macropinocytosis [14]; (2) GRP75 interacting with dynein light chain is involved in membrane-associated trafficking [41]. As a Pak1-interacting substrate, dynein light chain phosphorylation controls macropinocytosis [54]. Therefore, phosphorylation-modified GRP75 may significantly modulate this process; (3) we have demonstrated that GRP75-KD or inhibition significantly reduced Rac1 activation [34]. Since Rac1 activation always induces membrane ruffling and macropinocytic cup formation [32], dynamic expression and phosphorylation of GRP75 from S- to M-phase modulate macropinocytosis is logical (Fig. 8E).
GRP75 promoted centrosome duplication via recruiting Mps1 to centrosome
Dual-specific protein kinase MPS1 controls a number of steps in cell-cycle. Its activation promotes centrosome duplication but inhibits mitotic checkpoint response [56]. The finding of co-enrichment of MPS1 with GRP75 during cell-cycle (Fig. 3A, B) promoted us to explore whether centrosome duplication was regulated by GRP75. Confocal checking the asynchronously growing Cos7 and Skov3 cells showed that GRP75 did not bind with unduplicated centrosomes during cell-cycle, but bound with partial fraction of duplicated centrosomes during cell-cycle (Fig. 5A-F, Additional file 2: Fig. S9A-F). Next, the association of GRP75 with duplicated centrosomes from S- to M-phase in synchronously growing cells was observed by microscopy examination (Fig. 5G–K, Additional file 2: Fig. S9G-K). Since total expression level of GRP75 through the cell-cycle was at a similar level (Fig. 3B), phase-dependent association of GRP75 with duplicated centrosomes promoted us to further explore whether its expression or modification affects centrosome duplication. In hydroxyurea (HU)-induced centrosome re-duplication assays (Fig. 5L–N, Additional file 2: Fig. S9L-M), GRP75-OE significantly increased the frequency of centrosome amplification, and GRP75-KD markedly reduced the centrosome amplification. Notably, more increased centrosome amplification was detected in GRP75 phosphorylation-activation (T62D/S65D) cells, while less centrosome amplification was found in GRP75 phosphorylation-inactivation (T62A/S65A) cells (Fig. 5P, Additional file 2: Fig. S9O). Similar changes of centrosome amplification were found in asynchronously growing cells upon modulation of GRP75-expression level (Fig. 5O, Additional file 2: Fig. S9N). These results suggest that high-expression or phosphorylated activation of GRP75 is required for centrosome duplication.
As chaperone protein complexed with mitotic kinases can modulate their activity during the progression of cell-cycle [57, 58], we next tested whether centrosome-targeting of MPS1 was dependent on the presence of GRP75. Confocal checking the association of GRP75 and MPS1 at centrosome showed that these two targets were unable to simultaneously localize at centrosomes in GRP75-KD and phosphorylation-inactivation (T62A/S65A) cell. In contrast, significant centrosomal co-localization of GRP75 with MPS1 was detected in GRP75-OE and phosphorylation-activation (T62D/S65D) cells (Fig. 6A–F, Additional file 2: Fig. S10A-F). Further checking the centrosome-resident MPS1 in isolated fractions confirmed that GRP75-OE or phosphorylation-activation markedly increased MPS1 level, while GRP75-KD or phosphorylation-inactivation substantially reduced MPS1 level at centrosome (Fig. 6G, H, Additional file 2: Fig. S10G). These results suggest that centrosome-recruited MPS1 was promoted by high-expression or phosphorylation of GRP75.
Moonlighting protein has unrelated functions depending on the dynamic cellular context, which provides a connection between distinct biochemical processes [52, 59]. We have found that GRP75 is functionally enriched in heparan sulfate proteoglycan (HSPG)-mediated and membrane raft-associated endocytosis vesicles [26]. It can moonlight as a cell-cycle controller and endocytosis regulator [24, 27, 28]. Here, in OC cells, we also observed that GRP75-depletion induced cell-cycle G1-phase accumulation and GRP75-over-expression induced M-phase enrichment (Fig. 3E), reinforcing its role on cell-cycle control. Previous studies reported that GRP75 might control cell-cycle progress via cyclin-dependent kinas/TP53/Rb signaling [54, 60]. Our present data show that GRP75 associated preferentially with duplicating centrosomes (Fig. 5, Additional file 2: Fig.S9) and its phosphorylation was critically required for Mps1-translocating to centrosome (Fig. 6). Since MPS1 control centrosome duplication and mitotic checkpoint response and phosphorylated GRP75 super-activates Mps1 in a feedback manner [55, 56], the interdependence of GRP75 with MPS1 on centrosome-targeting (Fig. 6, Additional file 2: Figs. S10, S11) suggests that GRP75 is also required for centrosome duplication and mitotic checkpoint response. Additionally, centrosome is a highly unstable macromolecular complex and Hsp70–Hsp90 chaperone machinery is essential to maintain centrosome integrity for cell-cycle progression [57, 58]. Thus, centrosome-recruiting of GRP75 from S- to M-phase (Figs. 5, 6) may well help its maturation and accurate assembly of the bipolar spindle.
GRP75-driven, cell-cycle-dependent macropinocytosis in nanoparticle therapy
To explore whether GRP75-driven, cell-cycle-dependent macropinocytosis contribute to the uptake of Tat/pDNA-Ca2+ nanoparticles in vivo, inhibitors of GRP75, cell-cycle and macropinocytosis were applied to modulate the delivery of Tat/pDNA-Ca2+ nanoparticles in animals. NOD/SCID mice were subcutaneously transplanted with Skov3 cells labeled with DF (double fusion, Fluc-eGFP) reporter gene, and Tat/TF (triple fusion, RFP-Rluc-HSV-ttk)-Ca2+ nanoparticles were introduced by tail vein injection for suicide gene therapy (Fig. 7A, B). Bioluminescence imaging showed that, with GCV administration to induce suicidal cell-killing, Fluc signal from all groups with Tat/TF-Ca2+ therapy increased slightly until d15, indicating efficient tumor-suppression by the nanoparticles. When macropinocytosis or GRP75 inhibitor was intratumoral injected, Fluc signal from EIPA and MKT077 groups significantly stronger than that of the PBS group (Fig. 7C, D). In parallel groups with intratumoral injection of cell-cycle inhibitors, Fluc signal from GCB- and PTX-groups sharply declined, indicating more extensively regression of OC growth than nanoparticle mono-therapy. In contrast, significantly higher Fluc signal from lovastatin- and Ro3306-groups were observed compared to the nanoparticle mono-therapy group (Fig. 7C, D). Because cell-cycle inhibitors distinctly affected OC regression by Tat/TF-Ca2+ nanoparticles, we tried additionally intratumoral injection with EIPA in the four test groups. Comparing to the increased lovastatin- or Ro3306-induced Fluc signal, combined treatment with EIPA further markedly enhanced the Fluc signal, indicating decreased nanoparticle-therapy effect, due to the reduced macropinocytosis at cell-cycle G1- or G2-phase. Notably, combining EIPA with GCB or PTX brought reversed Fluc signal when compared to mono-treatment with GCB or PTX, indicating enhanced nanoparticle-therapy effect at S- or M-phase was due to active macropinocytosis at these two cell-cycle sub-phases (Fig. 7C, D). Furthermore, when MKT077 and EIPA were combined for intratumoral injection, the therapy effect of Tat/pDNA-Ca2+ nanoparticles was largely attenuated (Fig. 7C, D). We also checked body weight of treated mice, and no significant changes were observed in all experimental animals (Fig. 7E). TUNEL assays showed that treatment with Tat/TF-Ca2+ nanoparticle alone induced markedly apoptosis in OC tissues. More significant apoptosis was detected in the group with additional GCB- and PTX-treatments, and increased apoptosis was eliminated when EIPA was co-applied (Fig. 7F, Additional file 2: Fig. S12). These data strongly suggest that Tat/TF-Ca2+ nanoparticle-based suicide gene therapies were dominantly controlled by GRP75-driven, cell-cycle-dependent macropinocytosis.
To further determine the role of GRP75-driven, cell-cycle-dependent macropinocytosis on delivery of Tat/TF-Ca2+ nanoparticles, RLuc signal from TF plasmid was also investigated. As expected, Tat/TF-Ca2+ nanoparticles were specifically accumulated at OC site (Fig. 8A, B). When EIPA or MKT077 were further introduced, the OC-located RLuc signal from nanoparticles was sharply decreased, and was almost diminished after the co-injection of the two inhibitors. Weaker OC-targeted RLuc signal was observed after intratumoral co-injection with lovastatin or Ro3306, whereas further co-injection with GCB or PTX brought stronger RLuc signal at OC site (Fig. 8A, B). Again, when EIPA was introduced together with these cell-cycle inhibitors, synergistic TF RLuc signal was observed in lovastatin- or Ro3306-injected mice, and reversed TF RLuc signal were observed in GCB- or PTX-injected mice (Fig. 8A, B). The contrasted targeting of Tat/TF-Ca2+ nanoparticles to OC and distinct tumor-regression effect caused by different inhibitors was further confirmed by ex vivo RFP imaging and histological staining of RFP expression on resected OC tissues (Fig. 8C, D). Noticeably, no visceral toxicity was observed in OC-bearing mice treated by Tat/TF-Ca2+ nanoparticles with/without aforementioned inhibitors (Additional file 2: Fig. S13). Together, these results suggest that GRP75-driven, cell-cycle-dependent macropinocytosis dominantly underlies Tat/TF-Ca2+ nanoparticle-based suicide gene therapy in OC (Fig. 8E).