English

• The intracellular delivery of proteins is a promising strategy for definite regulation of cellular functions and accurate treatment of various diseases via directly targeting on intracellular sites [1–6], which avoids the limitations of small-molecule or genetic therapeutics such as strong side effects or random genetic alteration, off-target effects. Nowadays, therapeutic proteins occupy a huge portion in the pharmaceutical market [7–9]. However, all the clinically available proteins act on extracellular targets, as native proteins generally display limited membrane permeability and poor proteolytic stability [10–12]. Therefore, the cytosolic protein delivery carriers and technologies highly need to be developed for extending clinical application of proteins on intracellular targets. Several approaches have been developed to achieve the cytosolic delivery of proteins. Physical electroporation and microinjection methods are used to deliver proteins into the cytosol through transient disruption cell membrane, but these methods rely on specific instruments and may cause certain damage to cells [13,14]. Covalent modification of proteins with cell-penetrant peptides is also used to intracellular transduction of proteins through the enhancement of membrane penetrability, however, this approach requires synthetic and purification processes, and possibly decreases the proteins bioactivity [15–17]. Supramolecular self-assembly of carriers and proteins is the most promising and facile strategy in recent years [7–12]. The carriers for this approach generally include well-designed polymers and inorganic nanoparticles. For example, Cheng and coworkers exploited dendrimers functionalized with a series of functional groups, including boronic acid, guanidinium and dipicolylamine/zinc ion complex, to intracellularly deliver proteins [18–20]. Tew et al. reported cell-penetrating peptide mimetic polymers with rational guanidyl and hydrophobic segments rate to deliver proteins into cells [21–23]. Aida et al. reported a dendritic molecular glue, which is functionalized with multiple guanidinium pendants to adhere proteins, for cytosolic delivery of caspase-3 [24]. Rotello et al. used gold nanoparticles decorated with arginine to promote cytosolic delivery of proteins [25,26]. Most of the formerly reported protein carriers are based on elegantly designed polymers and nanomaterials. However, it remains a great challenge to intracellularly deliver proteins with different sizes and isoelectric points through macrocyclic molecule based noncovalent protein carriers [27,28].

  Pillararenes, as an emerging class of macrocyclic hosts, can be facilely functionalized to develop functional materials for different applications due to their highly symmetric pillar-like skeleton and π-rich cavities [29–34]. In our recent work, we reported that the densely preorganized guanidiniums by macrocyclic pillar[5]arene skeleton could facilitate intracellular delivery of native proteins [35]. From the previous work, we presume that the macrocyclic pillar[5]arene skeleton could preorganize functional groups to facilitate intracellular delivery of native proteins. However, the preorganization effect of pillar[5]arene skeleton for intracellular protein delivery is not proved using other functional groups. Amidiniums are well known to form stable salt bridges with carbonate or phosphate through charge-assisted hydrogen bonds in aqueous solutions [36,37]. Herein, we developed a new small molecular carrier based on amidinium functionalized pillar[5]arene (AP5) for facilitating delivery of proteins with different sizes and isoelectric points into cells (Scheme 1). As the amidinium groups are densely preorganized on pillar[5]arene skeleton, AP5 could not only glue proteins together to form AP5@protein complex through multiple salt-bridges between amidiniums and carboxyl residues on protein, but also promote AP5@protein complex to approach cell surface and further uptake by cell. Eight cargo proteins with different sizes and isoelectric points were delivered into the cytosol by AP5, and their bioactivities were well-maintained. This study provides a novel and versatile intracellular protein delivery carrier through the preorganization of amidiniums on the pillar[5]arene skeleton.

  Scheme 1

  

  Scheme 1.  Illustration of AP5 glues proteins with different sizes and pIs for intracellular delivery.

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  AP5 was synthesized by using 2-iminothiolane (Traust's reagent) and primary amine perfunctionalized pillar[5]arene as starting materials (Scheme S1 in Supporting information), and characterized by nuclear magnetic resonance spectroscopy (Fig. S1 in Supporting information). To investigate the cytosolic protein delivery capability of AP5, bovine serum albumin labelled by fluorescein isothiocyanate (FITC-BSA) was firstly selected as a model cargo protein. The self-assembly behaviour between AP5 and BSA was firstly studied through multiple methods. Upon titration the solution of FITC-BSA with AP5, the maximum fluorescence of FITC-BSA at 523 nm was gradually quenched and approached to a minimum when the molar ratio of [AP5]/[FITC-BSA] was 5:1 (Fig. 1a), which is likely because the microenvironment of FITC changed due to the adhesion of AP5 to FITC-BSA [38]. Dynamic light scattering (DLS) measurements disclosed that the mixture of AP5 and BSA at the ratio of 10:1 ([AP5]/[BSA], [BSA] = 1 µmol/L) yielded nanoaggregates with an average diameter of 160 nm, which was much larger than the size of the BSA in solution (6.6 nm, Fig. 1b), indicating that dozens of BSA were adhered together by AP5 to form nanoaggregates. The diameters of AP5@BSA complex gradually increased with increasing the molar ratio of [AP5] and [BSA], and the extremely high molar ratio influenced the stability of [AP5]@[BSA] complex (Fig. S2 in Supporting information). The nanoaggregates at the ratio of 10:1 ([AP5]/[BSA], [BSA] = 1 µmol/L) showed spherical morphology with a diameter of 105 nm on the transmission electron microscopy (TEM, Fig. 1c), implying that the mixture of BSA and AP5 self-assembled into nanoparticles. When the mixing molar ratio of [AP5]/[BSA] was increased from 0 to 15:1 (Fig. S3 in Supporting information), the surface zeta-potential of the AP5@BSA complex increased from negative to positive, indicating that amidinium moieties were also attached on the surface of the AP5@BSA complex. Moreover, circular dichroism (CD) spectra disclosed that there was no discernible alteration in the characteristic spectrum of BSA upon AP5 binding to BSA, suggesting the well-preserved secondary structure of BSA (Fig. 1d). This result implied that the adhesion of AP5 had minimal impact on the native structure of the protein, which is crucial for its biological activity.

  Figure 1

  

  Figure 1.  (a) Fluorescence changes of FITC-BSA (1 µmol/L) upon being titrated by AP5 (increasing AP5 concentration from 0.5 µmol/L to 5 µmol/L). The inset is fluorescence changes at 523 nm depending on [AP5]/[FITC-BSA].) (b) DLS data of free BSA (1 µmol/L, dark) and the mixture of BAS and AP5 ([BSA] = 1 µmol/L, [AP5] = 10 µmol/L, red) in H₂O at 25 ℃. (c) TEM image of AP5@BSA complexes ([AP5]/[BSA] = 10:1). (d) CD spectra of free BSA (1 µmol/L) and the mixture of BAS and AP5 ([BSA] = 1 µmol/L, [AP5] = 10 µmol/L, red) in H₂O.

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  As AP5 could adhere protein together to form nanoaggregates, the cytosolic protein delivery capability of AP5@FITC-BSA complexes on HeLa cells was studied. Interestingly, obvious green fluoresce of FITC-BSA was observed in the HeLa cells after being treated by AP5@FITC-BSA complexes for 4 h (Fig. 2a), which was in sharp contrast to the negligible cellular uptake of FITC-BSA alone. The cellular internalization of FITC-BSA at different mixing molar ratios of [AP5]/[FITC-BSA] from 0 to 15:1 was further studied by flow cytometry to screen the optimal mixing molar ratio for cytosolic delivery proteins. The [AP5]/[FITC-BSA] = 10:1 was chosen as the optimal mixing molar ratio for cytosolic delivery of proteins, as above 96% of cells were transduced at this mixing molar ratio within 4 h (Fig. 2b and Fig. S4 in Supporting information). And the [AP5]@[FITC-BSA] complex was also relatively stable in cell culture media containing 10% fetal bovine serum (Fig. S5 in Supporting information). The three-dimensional CLSM showed that FITC-BSA distributed in the cytoplasm and surrounded the nucleus, demonstrating the efficient transduction of FITC-BSA into the cells (Fig. 2c and Fig. S6 in Supporting information). Both AP5 and AP5@BSA at the working concentration for protein transduction exhibited negligible cytotoxicity to HeLa cells (Fig. S7 in Supporting information). The cellular internalization pathways of AP5@FITC-BSA complex were further studied using inhibitors for the reported internalization pathways. The cellular internalization of AP5@FITC-BSA by HeLa cells obviously decreased at the low temperature (at 4 ℃) or after being treated by chlorpromazine (a clathrin-mediated endosytosis inhibitor), and were not influenced by inhibitors for lipid-raft-dependent pathway (methyl-β-cyclodextrin, MβCD), micropinocytosis (amiloride), or caveolae-mediated endosytosis (genistein) [39–41], implying that AP5@FITC-BSA was uptaken into cells through clathrin-mediated endosytosis (Figs. 2d-f). After incubation for 6 h, the internalized AP5@FITC-BSA complex escaped from endosomes, and the proteins were released into the cytoplasm from the AP5@FITC-BSA complex (Fig. S8 in Supporting information), which is likely impelled by competitive interaction between various intracellular polyanionic species and AP5.

  Figure 2

  

  Figure 2.  (a) CLSM of HeLa cells treated with AP5@FITC-BSA ([AP5] = 10 µmol/L, [FITC-BSA] = 1 µmol/L) and free FITC-BSA (1 µmol/L) for 4 h. The scale bar is 40 µm. (b) The FITC-BSA transduction rate of HeLa cells treated by the AP5@BSA complexes at different molar ratio of FITC-BSA and AP5 ([FITC-BSA]/[AP5] = 1:2.5, 1:5, 1:10 and 1:15, [FITC-BSA] = 1 µmol/L) for 0.5, 1, 3 and 4 h, respectively. (c) Maximum intensity projection images of Z stacks (left) and 3D reconstructions (right) of AP5@FITC-BSA in HeLa cells. (120° z-axis rotation). The scale bar is 10 µm. (d, e) The relative fluorescence intensity of HeLa cells after being treated by various endocytosis inhibitors and AP5@FITC-BSA for 4 h. (f) CLSM of HeLa cells after being treated by various endocytosis inhibitors and AP5@FITC-BSA for 4 h. The scale bar is 40 µm.

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  To assess the robustness of AP5 in the process of protein delivery, we further investigated its capability to deliver proteins with different pIs and molecular weights, and into different cell lines. The negatively charged green fluorescent protein (GFP, pI = 6.2, Mw = 26.9 kDa), and positively charged FITC-labeled proteins, including IgG (from normal rabbit serum, pI = 10.3, Mw = 150 kDa), cytochrome C (Cyt C, pI = 10.5, Mw = 12.4 kDa), trypsin (pI = 10.5, Mw = 24.0 kDa, ), and ribonuclease A (RNase A, pI = 8.6, Mw = 13.7 kDa) were chosen as typical proteins for study the robustness of AP5 for cytosolic delivery of proteins. As shown in Fig. 3b, GFP was efficiently delivered into HeLa cells, and green fluorescence distributed in the cytosol after 6 h of incubation with AP5@GFP complexes at [AP5]/[protein] = 10/1. Similarly, other proteins labeled with FITC (including trypsin, IgG, Cyt C and RNase A) were also delivered into HeLa cells (Fig. 3b). Furthermore, other cell lines including 143B, A549, HUVEC, MCF-7 and MAD-MB-231 can all be transduced by AP5 with the FITC-BSA (Fig. 3c), indicating the robustness of AP5 in cytosolic delivery of membrane-impermeable proteins into different cell lines.

  Figure 3

  

  Figure 3.  (a) Confocal images of HeLa cells treated by AP5@FITC-BSA complexes for different time periods (2, 4, 6, and 8 h). The acidic compartments were stained by LysoTracker (red) ([FITC-BSA] = 1 µmol/L, [AP5] = 10 µmol/L). The scale bar is 40 µm. (b) CLSM of HeLa cells treated by various AP5@proteins complexes at the molar ratio of [protein]/[AP5] = 1:10 ([AP5] = 10 µmol/L) for 6 h. The scale bar is 40 µm. (c) CLSM of various cells treated by AP5@FITC-BSA complexe for 6 h ([FITC-BSA] = 1 µmol/L, [AP5] = 10 µmol/L). The scale bar is 40 µm.

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  In addition to its outstanding intracellular delivery capability, the delivered proteins retaining excellent biological activity are crucial for assessing the superiority of protein delivery carriers. Therefore, both horseradish peroxidase (HRP, pI = 7.2, Mw = 40.0 kDa, ) and β-galactosidase (β-Gal, pI = 5.0, Mw = 430 kDa) were chosen as two mode proteins to study whether the bioactivity of proteins is preserved after being delivered into the cells by AP5. In the presence of H₂O₂, the colorless substrate tetramethylbenzidine (TMB) can be catalyzed into blue products by HRP [42,43]. In the presence of TMB and H₂O₂, the cells treated by AP5@HRP exhibited distinct blue color (Figs. 4a and b), indicating the HRP activity was well kept after being delivered into the cell. About 90% of HRP activity was kept after being delivered into the cells (Fig. 4b). In the presence of H₂O₂, the nonfluorescent Amplex Red can also be catalyzed into red fluorescent resorufin. The well-preserved HRP activity was further confirmed by remarkable red florescence of resorufin in the AP5@HRP treated cell (Fig. 4c). In addition, the bioactivity of β-Gal was investigated after being delivered into the cells. Under the β-Gal catalysis, the colorless substrate 5–bromo-4–chloro-3-indolyl-β-D-galactoside (X-Gal) can be turned into a dark blue dye [42,43]. The cells treated by AP5@β-Gal showed an obvious dark blue color within the cells, while the cells treated by free β-Gal did not show any blue color. This result indicated that the β-Gal activity was well kept after being delivered into the cell (Figs. 4d and e). About 85% of β-Gal activity was preserved after being delivered into the cell by AP5 (Fig. 4f). These data indicate that the bioactivity of proteins delivered by AP5 was well maintained.

  Figure 4

  

  Figure 4.  (a) Illustration of HRP catalysis of TMB and Amplex Red. (b) HRP enzymatic activity for cells with different treatment (1: cells transduced by AP@HRP and added 50 µmol/L H₂O₂; 2: cells only transduced by AP@HRP; 3: cells terated by HRP and H₂O₂; 4: cells only terated by HRP) via TMB assays. (c) CLSM of HeLa cells treated with AP5@HRP and Amplex Red ([AP5] = 10 µmol/L, [HRP] = 1 µmol/L). The scale bar is 40 µm. (d) Illustration of β-gal catalysis of X-Gal. (e) The cells treated by free β-Gal and AP5@β-Gal for 4 h with a X-Gal stanning ([AP5] = 10 µmol/L, [β-Gal] = 1 µmol/L). (f) β-Gal enzymatic activity of the transduced cell via β-Gal enzyme activity assay kit.

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  Cytotoxic proteins play a crucial role in regulating cellular fate. The cytosolic delivery efficiencies of AP5 for toxic proteins, including trypsin, RNase A, Cyt C and saporin (Mw = 32.8 kDa, pI = 9.3), were further studied. Trypsin is a cell membrane-impermeable hydrolase, which can hydrolyze proteins and cause cell dysfunction [44]. Compared with the cells that were treated with trypsin alone, the cells treated with AP5@trypsin showed notable toxicity to the cells (Figs. 5a and b), indicating that intracellularly delivered trypsin by AP5 maintains robust protein hydrolytic activity. RNase A is an endonuclease, which catalyzes the hydrolysis of RNA strands and induces toxicity to cells [45,46]. Compared with the cells treated by free RNase A, the cells treated by AP@RNase A died significantly (Fig. 5c), implying that the intracellularly delivered proteins retained high hydrolysis activities to RNA strands and cause cell death. Cyt c is a membrane-impermeable mitochondrial heme protein, which causes programmed cell death through activating caspase-mediated apoptosis cascade [47,48]. As depicted in Figs. 5d and e, the AP5@Cyt C treated cells exhibited notably higher rates of apoptosis compared to those treated solely with Cyt C or the blank control. This indicated the effective intracellular delivery of Cyt C by AP5, resulting in the induction of cellular apoptosis, with a concentration-dependent response. Saporin is reported as a membrane-impermeable ribosome-inactivating protein, which can selectively block protein synthesis within mammalian cells through the depurination of a definite nucleotide [49,50]. The AP5@saporin complexes showed significant toxicity toward HeLa cells with an IC₅₀ of 102 nmol/L. However, saporin alone showed minimal toxicity on HeLa cells due to membrane-impermeability of saporin (Fig. 5f).

  Figure 5

  

  Figure 5.  (a) Illustration of therapeutic actions of trypsin, RNase A, Cyt C, and saporin after being delivered into cells. Cell viabilities of HeLa cells treated by (b) free RNase A and AP5@RNase A complexes, and (c) free Trypsin and AP5@Trypsin complexes, respectively. (d, e) Apoptosis rates of HeLa cell after incubated with free Cyt C and AP5@Cyt C for 4 h and further incubation for 48 h, respectively. (f) Cell viability of HeLa cells treated with saporin and AP5@saporin complexes. (g) Illustration of therapeutic actions of anti-Akt after being delivered into cells. (h) Cell viability of MCF-7 cells treated by free anti-pAkt and AP5@anti-pAkt at different anti-pAkt concentration for 24 h. [anti-pAkt]/[AP5] = 1/10. (i) Detection of caspase 3/7 in MCF7 cells after treated by free anti-pAkt or AP5@anti-pAkt for 6 h using a green dye labeled caspase substrate.

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  Furthermore, AP5 was applied for the intracellular delivery of antibody. Protein kinase B (Akt), as an intracellular signal transduction protein, is responsible for nuclear factor-кB (NF-кB) activation and its nuclear translocation, and responsible for other proteins in Akt signaling pathway involving cellular growth [51–53]. Blocking this pathway leads to the degradation of NF-кB and activation of caspases, which activate the apoptosis pathway causing cell death (Fig. 5g). In comparison with the cells incubated with anti-Akt alone, the cells treated with AP5@anti-Akt showed significantly reduced viability (Fig. 5h), suggesting that inactivation of Akt by intracellularly delivered anti-Akt led to cell death. In cells treated with AP5@anti-Akt, further evidence of the activity of caspase 3/7 enzymes was provided, which control the apoptosis pathway. Compared to cells treated solely with anti-Akt, cells treated with AP5@anti-Akt exhibited stronger green fluorescence, indicating the activation of the apoptosis pathway by blocking the Akt pathway (Fig. 5i). In a word, these above results strongly evidence that AP5 can serve as an excellent, facile protein carrier for cytosolic delivery of broad proteins.

  In conclusion, we develop a novel macrocyclic molecule-based protein carrier for cytosolic delivery of various proteins with different sizes and isoelectric points through preorganization of amidinium moieties on the pillar[5]arene skeleton. The preorganized amidinium moieties on the AP5 skeleton could glue proteins together to form AP5@protein complex through multiple salt-bridges between amidiniums and carboxyl residues on protein, but also promote AP5@protein complex to approach cell surface via multiple salt-bridges interactions and further cellular internalization of the AP5@protein complex. This study provides new perceptions for the design and synthesis of novel cell membrane penetrating peptide mimetic molecules for intracellular delivery of proteins.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Shuwen Guo: Writing – original draft, Visualization, Data curation. Haipeng Xu: Methodology. Zijun Cheng: Conceptualization. Leyong Wang: Supervision. Peng Yang: Supervision. Ruibing Wang: Supervision, Conceptualization.

  Acknowledgments

  We thank the financial support from the National Natural Science Foundation of China (No. 22101310), Fundamental Research Funds for the Central Universities (No. GK202207013), and NJU International Fellowship program.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111022.

  
  
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  Scheme 1  Illustration of AP5 glues proteins with different sizes and pIs for intracellular delivery.

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  Figure 1  (a) Fluorescence changes of FITC-BSA (1 µmol/L) upon being titrated by AP5 (increasing AP5 concentration from 0.5 µmol/L to 5 µmol/L). The inset is fluorescence changes at 523 nm depending on [AP5]/[FITC-BSA].) (b) DLS data of free BSA (1 µmol/L, dark) and the mixture of BAS and AP5 ([BSA] = 1 µmol/L, [AP5] = 10 µmol/L, red) in H₂O at 25 ℃. (c) TEM image of AP5@BSA complexes ([AP5]/[BSA] = 10:1). (d) CD spectra of free BSA (1 µmol/L) and the mixture of BAS and AP5 ([BSA] = 1 µmol/L, [AP5] = 10 µmol/L, red) in H₂O.

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  Figure 2  (a) CLSM of HeLa cells treated with AP5@FITC-BSA ([AP5] = 10 µmol/L, [FITC-BSA] = 1 µmol/L) and free FITC-BSA (1 µmol/L) for 4 h. The scale bar is 40 µm. (b) The FITC-BSA transduction rate of HeLa cells treated by the AP5@BSA complexes at different molar ratio of FITC-BSA and AP5 ([FITC-BSA]/[AP5] = 1:2.5, 1:5, 1:10 and 1:15, [FITC-BSA] = 1 µmol/L) for 0.5, 1, 3 and 4 h, respectively. (c) Maximum intensity projection images of Z stacks (left) and 3D reconstructions (right) of AP5@FITC-BSA in HeLa cells. (120° z-axis rotation). The scale bar is 10 µm. (d, e) The relative fluorescence intensity of HeLa cells after being treated by various endocytosis inhibitors and AP5@FITC-BSA for 4 h. (f) CLSM of HeLa cells after being treated by various endocytosis inhibitors and AP5@FITC-BSA for 4 h. The scale bar is 40 µm.

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  Figure 3  (a) Confocal images of HeLa cells treated by AP5@FITC-BSA complexes for different time periods (2, 4, 6, and 8 h). The acidic compartments were stained by LysoTracker (red) ([FITC-BSA] = 1 µmol/L, [AP5] = 10 µmol/L). The scale bar is 40 µm. (b) CLSM of HeLa cells treated by various AP5@proteins complexes at the molar ratio of [protein]/[AP5] = 1:10 ([AP5] = 10 µmol/L) for 6 h. The scale bar is 40 µm. (c) CLSM of various cells treated by AP5@FITC-BSA complexe for 6 h ([FITC-BSA] = 1 µmol/L, [AP5] = 10 µmol/L). The scale bar is 40 µm.

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  Figure 4  (a) Illustration of HRP catalysis of TMB and Amplex Red. (b) HRP enzymatic activity for cells with different treatment (1: cells transduced by AP@HRP and added 50 µmol/L H₂O₂; 2: cells only transduced by AP@HRP; 3: cells terated by HRP and H₂O₂; 4: cells only terated by HRP) via TMB assays. (c) CLSM of HeLa cells treated with AP5@HRP and Amplex Red ([AP5] = 10 µmol/L, [HRP] = 1 µmol/L). The scale bar is 40 µm. (d) Illustration of β-gal catalysis of X-Gal. (e) The cells treated by free β-Gal and AP5@β-Gal for 4 h with a X-Gal stanning ([AP5] = 10 µmol/L, [β-Gal] = 1 µmol/L). (f) β-Gal enzymatic activity of the transduced cell via β-Gal enzyme activity assay kit.

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  Figure 5  (a) Illustration of therapeutic actions of trypsin, RNase A, Cyt C, and saporin after being delivered into cells. Cell viabilities of HeLa cells treated by (b) free RNase A and AP5@RNase A complexes, and (c) free Trypsin and AP5@Trypsin complexes, respectively. (d, e) Apoptosis rates of HeLa cell after incubated with free Cyt C and AP5@Cyt C for 4 h and further incubation for 48 h, respectively. (f) Cell viability of HeLa cells treated with saporin and AP5@saporin complexes. (g) Illustration of therapeutic actions of anti-Akt after being delivered into cells. (h) Cell viability of MCF-7 cells treated by free anti-pAkt and AP5@anti-pAkt at different anti-pAkt concentration for 24 h. [anti-pAkt]/[AP5] = 1/10. (i) Detection of caspase 3/7 in MCF7 cells after treated by free anti-pAkt or AP5@anti-pAkt for 6 h using a green dye labeled caspase substrate.

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