Selective intracellular delivery of protein in cells with nanocarriers sensing cathepsin b activity

Abstract

The present invention provides a cathepsin B-sensitive probe comprising a conjugate of a cathepsin B-sensitive linker and a fluorescent emitter.

Description

DESCRIPTIONTitle of InventionSelective intracellular delivery of protein in cells with nanocarriers sensing cathepsin B ActivityTechnical FieldThe present invention relates to a cathepsin B-sensitive probe comprising a conjugate of a cathepsin B-sensitive linker and a fluorescent emitter. Further, the present invention relates to an intracellular protein delivery system using a cathepsin B-sensitive linker.All disclosures of the references cited herein are incorporated herein by reference in their entirety.Background ArtOvercoming the endo / lysosomal barrier is crucial for effective intracellular delivery of proteins1’1. Failing to escape from the endo / lysosomal compartments incapacitates the endocytosed proteins for working inside the cells and leads to their degradation12’31. Nanocarriers with endosomal escape capability, such as lipid14 6Jand polymer-based carriers17-91, and virus-like formulations110’11], are widely studied for their ability to break endo / lysosomes via pH-buffering, membrane destabilization, and fusogenic interactions. Such approaches are designed to powerfully disrupt the endo / lysosomes for driving the payloads into the cytosol as much as possible, though their endosomal escape behavior remains largely unspecific. In contrast, developing nanocarriers with selective endosomal escape in specific cells could offer a promising alternative for targeted intracellular protein delivery120].For attaining selective endosomal escape, the nanocarriers should be designed to activate their endosomal escape functions upon sensing stimuli that are specifically present in the endosomes of target cells. Thus, defining the proper stimulus to trigger the endosomal escape is essential for this approach. The acidity of the endo / lysosomal compartments has been applied as a stimulus for activating the endosomal escape of nanocarriers. However, this is a common feature wildly presented in endo / lysosomes12’]. On the other hand, several' enzymes are differentially expressed in the endo / lysosomal environments of particular cells, e.g., cancer cells1221, offering unique spurs for nanocarrier activation. Notably, the lysosomal cysteine protease cathepsin B (CTSB) is upregulated in the endo / lysosomes of cancer cells123,241, leading to the clinical application of tumor-targeted CTSB-sensitive prodrugs1251and antibody-drug conjugates (ADCs)126,271.Summary of InventionHowever, developing CTSB-sensitive systems for intracellular protein delivery is a challenging task, since proteins would be threatened by the enriched protease setting within the endo / lysosomal pathway, which suggests a spatiotemporal window of endosomal escape activation versus payload degradation. Therefore, careful investigation is necessary for revealing the performance of a CTSB-sensitive intracellular delivery system for proteins.The present invention is as follows.[1] A cathepsin B-sensitive probe comprising a conjugate of a cathepsin B-sensitive linker and a fluorescent emitter.[2] The probe according to [1], wherein the cathepsin B-sensitive linker comprises a valine-citrulline peptide.[3] The probe according to [2], wherein the linker containing the valine-citrulline peptide is azido- [polyethylene glycol)]3-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate.[4] Protease-sensitive probe comprising a conjugate of protease-sensitive protein and a fluorescent emitter.[5] A protein-probe complex comprising the probe according to [1] bound to a protein.[6] The complex according to [5], wherein the protein is bovine serum albumin.[7] A method for detecting a cell having a cathepsin B activity, comprising contacting the probe according to any one of [1] to [3] or the complex according to [5] or [6] with a test cell.[8] The method according to [7], wherein the cell having a cathepsin B activity is a cancer cell.[9] A method for detecting protease-rich cell, comprising contacting the probe according to [4] with a test cell.

[0010] An intracellular protein delivery system, comprising a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein.

[0011] A protein delivery kit into a cell, comprising a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein.

[0012] The system according to

[0010] , wherein the protein is an antibody.

[0013] The kit according to

[0011] , wherein the protein is an antibody.Brief Description of DrawingsFigure 1. Characterization of the probes and controlling formulations. A. Emission spectra of the probes and controlling formulations. Equivalent free dyes (Cy5 or A488) were used as standard. B. Fluorescence intensities of probes during incubation with lysosome lysates. Data are plotted as the mean ± S.D. (n = 3). C. Representative CLSMimages of 4T1 cells incubated with the probes and controlling formulations after 16 h. Scale bar = 10 pm.Figure 2. Screening of endo / lysosomal CTSB activity and protein degradation rate among cell lines. A. Representative confocal laser scanning microscopy images of cells incubated with Pcm and CCTSB. Red: activated PCTSB; green: CCTSB. Scale bar = 10 pm. B. Time-dependent changes of Fcy5 / FA488 in CT26 and Hela-Luc cells incubated with PCTSB and CCTSB- Data are plotted as the mean ± S.D. (n = 25). C. Screening of the CTSB activity in different cells indicated by the PCTSB activation fraction at different time points. D. Representative confocal laser scanning microscopy images of cells incubatedand CdeS. Red: activated Pdeg; green: Cdeg. Scale bar = 10 pm. E. Time-dependent change of Fcy5 / FA488 in CT26 and Hela-Luc cells incubated with P^and Cdeg. Data are plotted as the mean ± S.D. (n = 25). F. Screening of the protein degradation rate in different cells indicated by the Pdeg activation fraction at different time points.Figure 3. The correlation of CTSB activity with CTSB abundance or endo / lysosomal pH. A. Relative CTSB concentrations in lysates of whole cell (WC) and endo / lysosomes (ELs) measured by ELISA. Data are shown as the mean ± S.D. (n = 3). Black bars: Cancer cells; Grey bars: Non-cancerous cells. B. Correlation analysis between CTSB activity and CTSB abundance. C. Endo / lysosomal pH distribution of different cell lines. Data are plotted as the mean ± S.D. (n = 25). D. Average endo / lysosomal pH and optimal pH convergence index (OPCI) of different cell lines. Data are plotted as the mean ± S.D. (n = 25). E. Correlation analysis between CTSB activity and OPCI value.Figure 4. Characterization of anti-NPC / l / p. A. Average diffusion coefficient of native anti-NPC antibodies and anti-NPC / l / p measured by fluorescence correlation spectroscopy (FCS). Data are plotted as the mean ± S.D. (n = 3). B. Average hydrodynamic diameter of free anti-NPC antibodies and anti-NPC / l / p calculated from the diffusion coefficient of the samples. Data are plotted as the mean ± S.D. (n = 3). C. HPLC detection of native anti-NPC antibodies, anti-NPC / l / p and anti-NPC / l / p after incubation with CTSB. D. Decrease of the diffusion time in FCS measurement of anti-NPC / l / p after incubation with different CTSB concentration. Data are plotted as the mean ± S.D. (n = 3).Figure 5. CTSB-sensitive system realized cell-specific antibody delivery. A. Representative confocal laser scanning microscopy images revealing the delivery of anti- NPC into CT26 and Hela-Luc cells. Blue: nuclei; red: A647-labeled anti-NPC; green: Lysotracker. Scale bar = 10 pm. Co-localization coefficient were plotted in the bar graphs as the mean ± S.D. (n = 15). p values were calculated by One-Way ANOVA. B. Fluorescence intensity of the extracted nuclei from cells incubated with anti-NPC / l / p. The signal was normalized to negative controls where the cells were treated with free anti- NPC. Data are shown as the mean ± S.D. (n = 3). p values were calculated by One-WayANOVA. C. Delivery efficiency indicated by the normalized fluorescence intensity from extracted nuclei from different cells. Data are shown as the mean ± S.D. (n = 3). D. Correlation analysis between delivery efficiency and EACTSB at 4h.Figure 6. Design and activation mechanisms of the probes for tracking the CTSB activation and the controlling formulations for imaging protein degradation in cells.Figure 7. Design and mechanism of the CTSB -sensitive system attaining cell-specific intracellular antibody delivery via the selective endosomal escape process.Figure 8. A. Selective activation of Peras by different proteases. B. Activation rate of PCTSB under different CTSB concentrations. C. Activation rate of Pdeg under different CTSB concentrations. Data are plotted as the mean ± S.D. (n = 3). D. Co-localization coefficient of probes signals (red) to corresponding controlling formulations signals (green) after 16 h incubation in different cell lines. Data are plotted as the mean± S.D. (n = 3). E. Representative confocal images of 4T1 cells incubated with PCTSB or P^^red). The endo / lysosomes were stained with Lysotracker Green (green). Scale bar = 10 pm. F. Representative confocal images of Z-FA-FMK treated 4T1 cells incubated with PCTSB and CCTSB (upper panel) or Pdeg and Cdeg(lower panel). Red: Peras or P^ra green: CCTSB or Cdeg- Scale bar = 10 pm.Figure 9. Scheme showing the workflow for analyzing the images of cells incubated with the probes and controlling formulations to determine the activation rate of the probes.Figure 10. Representative CLSM images of different cells incubated with PCTSB and CCTSB (left panel) or Pdeg and C*g(right panel). Red: probes; green: controlling formulations; scale bar = 10 pm.Figure 11. CTSB activity and protein degradation rate in different components of endo / lysosomes system. A. Representative confocal laser scanning microscopy images of cells incubated with Peras, Pdeg or A647-BSA. Blue: nuclei; red: activated probes or A647- BSA; green: GFP-labeled early endosomes (EE), late endosomes (LE) and lysosomes (lyso). Scale bar = 10 pm. B. Co-localization analysis of A647-BSA with different components of endo / lysosomes system. C. Co-localization analysis of activated Peras with different components of endo / lysosomes system. D. Co-localization analysis of activated Psegwith different components of endo / lysosomes system. Data are shown as the mean ± S.D. (n = 10)Figure 12. Scheme showing the workflow of analyzing the images of the cells incubated with the pH-sensitive probe.Figure 13. Representative CLSM images of cells incubated 4 h with TMR-Dextran-Fluor.Red: TMR; green: Fluor. Scale bar = 20 pm.Figure 14. pH-dependance of CTSB activity. A. PCTSB was incubated with CTSB in PBS with different pH values. Fluorescence intensity of each solution was tracked to indicate the activation of PCTSB. B. The fluorescence intensity of each solution after 4 h incubation (dotted red square in A) was plotted to indicate the relative CTSB activity under the corresponding pH. The dotted line indicates the pH (=5.0) where CTSB showed highest activity. Data are plotted as the mean ± S.D. (n =3).Figure 15. Analysis of anti-NPC delivery efficiency to different cells. A. Representative CLSM images of different cells after incubation with anti-NPC / l / p for 24 h. Blue: Hoechst 33342; green: Lysotracker; red: anti-NPC; scale bar = 10 pm. B. The effective CTSB activity (EAcrss) of different cells at 4 h and 8 h. C. Correlation analysis between delivery efficiency and EACTSB at 8 h. D. Matrix showing the Pearson r values between every two parameters among delivery efficiency, EACZSB at 4 h and EACTSB at 8 h.Figure 16. The workflow of analyzing the images of the cells incubated with the pH- sensitive probe.Description of Embodiments1. OverviewThe present invention relates to a design of probes and the antibody-polymer conjugate drug delivery system. The present invention relates to a methodology of using the probes for quantifying the endo / lysosomal CTSB activity in living cells. Further, the present invention relates to a methodology of using the differentiated endo / lysosomal CTSB activity to control the endosomal escaping capability of drug delivery systems.The probes are easy to be manufactured and the protocol of using the probes for detecting the CTSB activity is simple and universally applicable under laboratory conditions. Thus, the probes can be applied as a commercial experimental kit for research usage. The formulation of the antibody-polymer conjugate system is simple and easy to be manufactured. Also, the components in the system, including the antibodies, the linker, and the polymer materials can be changed for realizing different purposes. This system is potentially applicable for clinical usage.In the present invention, an albumin-based fluorescent probe was firstly designed for detecting the enzymatic activity of CTSB in living cells. As a macromolecule, the probe can specifically and quantitatively reveal the dynamic change of CTSB activity level inthe endosomes and / or lysosomes (hereinafter referred to as "endo / lysosomes") in different cells along the endocytosis process. The results have confirmed that, in different cells, the endosomal and / or lysosomal (hereinafter referred to as "endo / lysosomal") CTSB activity profiles are highly-differentiated. Especially in some cancer cells, the endo / lysosomal CTSB activities are significantly higher than non-cancerous cells. Thus, the endo / lysosomal CTSB activity can be a cell-specific biomarker to control the conformation change of drug delivery systems. Based on these discoveries, a novel drug delivery system loaded with antibody was designed. The system has the CTSB-triggered endosomal escaping capability, and can realize a cell-specific intracellular delivery of antibodies by sensing the differentiated endo / lysosomal CTSB activity in different cells.The design of the probes is shown in Figure 6. In the CTSB-sensitive fluorescent probe (PCTSB), bovine serum albumin (BSA) was used as the protein substrate. A CTSB- cleavable linker, Azido-[poly(ethylene glycol)]3-valyl-citrullyl-(4-aminobenzyl)-(4- nitrophenyl) carbonate (Azido-PEG3-Val-Cit-PAB-PNP) was conjugated to the BSA through the reaction between PAB-PNP group and the amine groups on the BSA. Next, Cyanine-5-dibenzocyclooctyne (Cy5-DBCO) was reacted with the azide groups of the linkers. This modified BSA, referring as the PCTSB, had a high degree of labelling (DOL) by the Cy5 dyes (> 6 Cy5 dyes in one BSA molecule), which induced a strong selfquenching between the dyes to cause a limited fluorescent emission.If the PCTSB is confronted with CTSB-enriched envrionment, the Azido-PEG3-Val-Cit- PAB-PNP linker binding the Cy5 dyes with the BSA proteins can be cleaved by the enzyme, thus the Cy5 dyes can be released from the surface of BSA and relieve the selfquenching to regain the strong fluorescence emission.Therefore, the increasement of the fluorescence intensity from the probes can indicate the enzymatic activity of CTSB in the surrounding environment. In the case of quantitative analysis, an “always on” formulation (controlling formulation, referring as CCTSB) was introduced. In CCTSB, the linkers were conjugated to BSA through the same pattern in PCTSB, while a different dye, Alexa Fluor 488 (A488) was conjugated to the azide groups of the linkers, and the DOL of A488 was controlled to a lower level (<3). This low level of DOL could not induce the self-quenching between the fluorophores thus the fluorescence emission of the CCTSB was constant without responsing to CTSB. Therefore, the CCTSB can be used as an indicator to eveluate the cellular uptake of the probes. The design of the drug delivery system is shown in Figure 7.The Azido-PEG3-Val-Cit-PAB-PNP was conjugated to the antibodies. Next, a functional cationic polymer, poly(ethylene glycol)-poly{N-[N’-(2-amino-ethyl)-2- aminoethyl]aspartamide} (PEG-pAsp(DET)), was modified by DBCO-PEG4- succinimidyl Ester (DBCO-PEG4-NHS ester). The modified PEG-pAsp(DET) (PEG-pAsp(DET)-DBCO) was then conjugated to the azide groups of the linker to form the antibody-polymer conjugate system. The system modified with the Val-Cit linker can sense the endo / lysosomal CTSB activity in the cells.. In cells with high endo / lysosomal CTSB activity, the Val-Cit linker can be cleaved rapidly and the PEG-pAsp(DET) polymers can detach from the antibodies and contact with the endo / lysosomal membrane to destabilize the membrane structure. Thus, the endo / lysosomes can be broken by the polymers and the antibodies can escape from the endo / lysosomes and enter the cytosol. However, in cells with low endo / lysosomal CTSB activity, the antibody-polymer conjugate system remains intact and trapped in the endo / lysosomes until all the components are degraded by acidity and the proteases in the lysosomes.The fluorescent probe can realize a dynamic and specific evaluation of the endo / lysosomal CTSB activity in living cells with imaging methods. After incubating PCTSB and CCTSB with living cells, fluorescence signals could be detected from the cells (Figure 1). The red fluorescence signals, referring to the activated PCTSB, were localized in some spherical structures in the intracellular spaces, corresponding to the endo / lysosome compartments.Upon the increase of incubation time, the intensity of the red fluorescence elevated gradually, indicating the escalation of the accumulative enzymatic activity of CTSB in the endo / lysosomes along the process of endocytosis. By calculating the ratio of red fluorescence intensity to green fluorescence (FCy5 / FA488) in the endo / lysosomes, the endo / lysosomal CTSB activity level can be quantified in living cells.In different cells, the profile of endo / lysosomal CTSB activity showed significant difference. For example, in CT26 cells, high CTSB activity could be detected in the earlier time points (4-8h) during the incubation, while in Hela-Luc cells, the endo / lysosomal CTSB activity showed a much slower escalation. Thus, such differentiated CTSB profile in endo / lysosomes can help to realize a cell-specific intracellular delivery of antibodies by the invented antibody-polymer conjugate drug delivery system.For a proof-of-concept, anti-nuclear pore complex (anti-NPC) antibodies were used as a model in the drug delivery system. The endosomal escaping efficiency of the system was firstly evaluated in CT26 and Hela-Luc cells by imaging-based co-localization analysis (Figure 5A). In Hela-Luc cells, a higher co-localization between the red fluorescence (Cy5-labelled anti-NPC antibodies) and green fluorescence (Lysotracker staining) was found than that in the CT26 cells, indicating that the system has a lower endosomal escaping ability in Hela-Luc cells than in CT26 cells.Moreover, as the anti-NPC antibodies can bind to the nucleus membrane if they are successfully delivered into cytosol spaces, the fluorescence signal of the Cy5-labelledanti-NPC antibodies from the isolated nuclei of the cells can be used as a quantitative index of the antibody delivery efficiency (Figure 5B). In different cells, the antibody delivery efficiency by the system was highly differentiated, and the delivery efficiency was correlated with the endo / lysosomal activity profile detected by the fluorescent probes.The differential enzymatic activity in the endo / lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo / lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.2. Self-quenched fluorescent probesTo understand the spatiotemporal window of CTSB activation versus protein payload degradation, we first designed self-quenched fluorescent probes based on albumin for visualizing the cleavage of CTSB-sensitive linkers and the degradation of proteins along endocytosis. The design of the probes is shown in Figure 6. To construct the CTSB- sensitive probe (Peras), we used the CTSB-sensitive linker, azido- [poly (ethylene glycol)]3-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate (Azido-PEGs-Val- Cit-PAB-PNP), in which the valine-citrulline (Val-Cit) peptide is specifically cleavable by CTSB1281. We conjugated this linker to the amine residues of bovine serum albumin (BSA) via the reactive PNP moieties. Then, cyanine-5-dibenzocyclooctyne (Cy5-DBCO) was conjugated to the linker via click reaction between the DBCO and the azido groups.The high degree of Cy5 labeling to BSA lead to strong self-quenching between the fluorophores, which decreased the fluorescence emission. However, once the Peras encountered CTSB, the Val-Cit peptide of the linker was cleaved and the Cy5 molecules retrieved their fluorescence emission. Thus, the increment of the fluorescence emission of Peras was used as an indicator of the enzymatic CTSB activity. Moreover, we designed an “always-on” controlling formulation (Ccras) for following biological experiments. In Ccras, regulated amount of Alexa Fluor 488 (A488) molecules were conjugated to BSA via the same linker. The lower degree of A488 labeling to BSA was not enough for selfquenching, so the fluorescence signal of this formulation would not be sensitive to CTSB. For monitoring the rate of BSA degradation by the protease in endo / lysosome systems,another probe (P<feg) was designed, in which a high amount of Cy5 dyes was conjugated to BSA via amide bond formed from the condensation reaction between the amines on the protein and N-hydroxy succinimide (NHS) ester group of the dye. In P< / eg, the selfquenching effect between the Cy5 molecules was only retrieved after the BSA is degraded. Also, a controlling formulation (Cdeg) with constant fluorescence emission was constructed by conjugating a regulated amount of A488 NHS-ester to the amine residues of the BSA.3. A cathepsin B-responsive carrier for protein deliveryAlthough therapeutic proteins are expected to be promising in the treatment of intractable diseases, their systemic administration involves various problems including instability, short half-life, and non-specific immune reactions, etc. Thus, a protein delivery approach using stimuli-responsive nanocarriers may be an effective strategy to enhance protein activity in target tissues in a tissue selective manner. In the present invention, there have been developed a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein, with the aim of releasing the loaded proteins in a cathepsin B-dependent manner.Cationic polymer having a side chain containing a primary amineThe cathepsin B-responsive carrier for protein delivery to cells or tissues comprises a block copolymer represented by the following general formula (I) or (II):In the general formula (I), R11and R12each independently represent a hydrogen atom, or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms, or an azide, an amine, maleimide, a ligand or a labeling agent, R32represents methylene group or ethylene group.L11represents NH, CO, or a group represented by the following formula (11): -(CH2)PI-NH- (11)(wherein pl represents an integer of 1 to 6), or a group represented by the following formula (12):-L2a-(CH2)qi-L3a- (12)(wherein L2arepresents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L3arepresents NH or CO, and ql represents an integer of 1 to 6),R31and R33each independently represent a group having an amino group or a group having a linker (provided that, in at least one of each of the (ml 1+ml 2) monomer units, R31and / or R33has a linker).Specifically, R31and R33each independently represent general formula (41) or (42) below:-NH-(CH2)r-Xn(41)-(wherein, X11represents an amine compound residue derived from primary, secondary * or tertiary amine compound or quaternary ammonium salt, and r represents an integer of 0-5)-[NH-(CH2)si]ti -X12(42)(wherein, X12is synonymous with X11, and si and tl, independently from each other and independently between [NH-(CH2)SI] units, represent integers of 1-5 and 2-5,respectively),In the group having a linker, in the formulae (41) and (42), X11and X12are linkers.More specifically, R31and R33include, for example, -NH-NH2,(DBCO represents a dibenzocyclooctyne group.)Preferably, the group having an amino group isExamples of the group having a linker include a group represented by the formula:.More specifically, the DBCO is conjugated to the primary amine by an amide bond.Example of the group having the DBCO is represented by the following formula:However, the linker is not limited to DBCO, and may be BCN(bicyclo[6.1.0.]nonyne), and these linkers may be modified with PEG or the like. DBCO and BCN linkers may be commercially available (Funacosi). These linkers react with azide-labeled molecules or biomolecules. When an azide compound (see the Examples) is used as a cathepsin B- sensitive linker (cathepsin-cleavable linker) and is bound to a protein, for example, a protein or the like can be bound by using a DBCO.In the general formula (I), ml 1 and ml 2 each independently represent an integer of 0 to 500 (provided that the sum of ml 1 and ml2 represents an integer of 10 to 500). In one embodiment of the invention, ml 1 and ml2, each independently represent an integer of 1 to 500. In the general formula (I), ml 3 represents integer of 1 to 5, the symbol means that (mi l + ml 2) units of the respective monomer units shown on the left and right sides of this symbol may be in any sequence. For example, when a block moiety composed of monomer units A and B is represented by [-(A)a- / -(B)b-J, the symbolmeans that a units of A and b units of B, i.e., (a + b) units in total of the respective monomer units may be linked at random in any sequence (provided that all the monomer units A and B are linked in a linear fashion).At least one of the monomeric units of the (ml l+ml2) is a group in which R31and / or R33have a linker. For example, considering a block copolymer in which ml 1 is 5 and ml2 is composed of 5 monomers, in one embodiment, one of the monomers constituting mil has a linker, the monomer constituting ml 2 may be a block copolymer without a linker, in another embodiment, one of the monomers constituting mil may be a block copolymer having a linker and one of the monomers constituting ml2 may be a block copolymer having a linker, and in yet another embodiment, all five of the monomers constituting mil may be a block copolymer having a linker, and all five of the monomers constituting ml 2 may be a block copolymer having no linker.In the general formula (I), the block moiety whose number of repeating units (degree of polymerization) is nl corresponds to the PEG moiety, while the block moiety composed collectively of submoieties whose number of repeating units is ml 1 and ml 2, respectively (i.e., the moiety shown in brackets [ ] in general formula (I)) corresponds to the polycation moiety. More specifically, nl represents an integer of 1 to 500 (preferably 100 to 400, more preferably 200 to 300).In the general formulae (I) and (II), PEG moiety is linear. However, the PEG moiety may be branched. In one embodiment of the invention, the PEG moiety may have a 4-arm or 8 -arm structure.In the present invention, PEG-pAsp(DET) can preferably be used, as shown in Example.In the general formula (II), R21, R22, L21, n2, m21, m22 and m23 are synonymous with R11, R12, L11, nl, mil, ml2 and ml3, respectively. m24 and m25 each independently represent an integer of 1 to 5.The molecular weight (Mn) of the cationic polymer represented by general formulae (I) and (II) is not limited in any way, but it is preferably 23,000 to 45,000, and more preferably 28,000 to 34,000. With regard to the individual block moieties, the PEG moiety has a molecular weight (Mw) of preferably 8,000 to 15,000, and more preferably 10,000 to 12,000, while the polycation moiety as a whole has a molecular weight (Mn) of preferably 15,000 to 30,000, and more preferably 18,000 to 22,000.Examples of the above linear or branched alkyl group containing 1 to 12 carbon atoms include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, a tert-butyl group, a n-pentyl group, a n-hexyl group, a decyl group and an undecyl group, etc. Moreover, examples of substituents on the above alkyl group include an acetal-protected formyl group, a cyano group, a formyl group, a carboxyl group, an amino group, an alkoxycarbonyl group containing 1 to 6 carbon atoms, an acylamido group containing 2 to 7 carbon atoms, a siloxy group, a silylamino group, and a tri alky Isiloxy group (each alkylsiloxy group independently contains 1 to 6 carbon atoms), etc.A ligand molecule refers to a compound used with the aim of targeting a certain biomolecule, and examples include an antibody, an aptamer, a protein, an amino acid, a low molecular compound, a monomer of a biological macromolecule and so on. Examples of a fluorescent emitter include, but are not limited to, fluorescent labeling agents such as a rare earth fluorescent labeling agent, Alexa, cyanine, coumarin, dimethylaminosulfonyl benzoxadiazole (DBD), dansyl, nitrobenzoxadiazole (NBD), pyrene, fluorescein, a fluorescent protein and so on.When the above substituent is an acetal-protected formyl group, this substituent can be converted into another substituent, i.e., a formyl group (or an aldehyde group; -CHO) upon hydrolysis under acidic mild conditions. Moreover, when the above substituent (particularly on R11) is a formyl group or is a carboxyl group or an amino group, for example, an antibody or a fragment thereof or other functional or targeting proteins may be linked via- these groups.4. ProteinsThe protein to be used in the present invention may be of any type, as long as it is originally among basic or neutral proteins. The protein to be used in the presentinvention encompasses not only simple proteins, but also glycoproteins and lipoproteins, etc. Moreover, the protein to be used in the present invention is not limited to those consisting of full-length amino acid sequences, and also encompasses their partial fragments and peptides, etc., as well as proteins consisting of two molecules (dimer) or more molecules, and fusion proteins formed between partial or full-length sequences thereof. Moreover, the protein to be used in the present invention is not limited to those composed of natural amino acids, and also encompasses modified proteins comprising at least some unnatural amino acids as constituent members. Furthermore, the protein to be used in the present invention also encompasses those modified as appropriate to have various labeling substances or the like, if necessary. Specific examples of the protein to be used in the present invention include, but are not limited to, heme proteins, various cytokines, various enzymes, or antibodies (e.g., antibodies against nuclear pore complexes) or antibody fragments, etc.5. Method for detecting a cell having a cathepsin B activity, or a protease-rich cell.The probes of the invention can be used as reagents to detect cells with cathepsin B activity or protease-rich cells.A method for detecting cells having cathepsin B activity or protease-rich cells includes, for example, (a) reacting the probe of the present invention with a test cell, and (b) detecting a label emitted from the probe, include.To detect the signal emitted from the label after the reaction, for example, measure the luminescence intensity when the quenched probe emits light due to the activity of cathepsin B or the action of protease (see Fig. 6).6. Device and Kit for a protein deliveryThe protein delivery kit and device of the present invention is characterized by comprising the above a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein. This kit can be preferably used, for example, in various therapies using a desired protein (e.g., enzyme replacement therapy, antibodybased immunotherapy).In the kit of the present invention, the cationic polymer may be stored in any state, and a solution or powder state may be selected in consideration of its stability (storage quality) and easiness of use, etc. The kit of the present invention may further comprise other components, in addition to the above complex. Examples of other components include various buffers, various proteins to be introduced into cells, dissolution buffers, and instructions for use (instruction manual), etc.Methods and conditions are the same as described above. Moreover, examples of thevarious diseases include, but not limited to, cancers (for example, lung cancer, pancreas cancer, brain tumor, liver cancer, breast cancer, colorectal cancer, neuroblastoma and bladder cancer), cardiovascular diseases, musculoskeletal diseases and central nervous system diseases.The above-described pharmaceutical composition can be formulated by an ordinary method by suitably selecting and using a diluent, a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a lubricant, a surfactant, a dispersant, a buffer, a preservative, a solubilizing aid, an antiseptic, a flavoring agent, a soothing agent, a stabilizer, a tonicity adjusting agent and the like which are generally used for drug production. In addition, an intravenously injectable agent (including intravenous drip) is usually employed as the form of the pharmaceutical composition. For example, the pharmaceutical composition may be provided in a single-dose ampoule or in a multiple-dose container. cells.EXAMPLESThe present invention will be further described in more detail by way of the following illustrative examples, which are not intended to limit the scope of the invention.[Example 1]1. Materials and Methods1.1. MaterialsAzido- [poly (ethylene glycol)]3-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate (Azido-PEGs-Val-Cit-PAB-PNP) was purchased from AxisPharm (San Diego, USA). Bovine serum albumin (BSA) was purchased from Fujifilm (Tokyo, Japan). Cyanine5-dibenzocyclooctyne (Cy5-DBCO), Cyanine5 N-hydroxy succinimide ester (Cy5-NHS ester), human cathepsin B (CTSB), human cathepsin L (CTSL), human matrix metalloproteinase- 1 (MMP-1), human matrix metalloproteinase-2 (MMP-2) were purchase from Sigma-Aldrich (St. Louis, USA). Alexa Fluor 488-dibenzocyclooctyne (A488-DBCO), Alexa Fluor 488 N-hydroxy succinimide ester (A488-NHS ester), BCA assay kit, trypsin, protease inhibitor cocktail, CellLight Early Endosome-GFP, CellLight Late Endosome-GFP, CellLight Lysosome-GFP, Alexa Fluor 647 conjugated BSA (A647- BSA), Tetramethylrhodamine and fluorescein labeled dextran (TMR-Dextran-Fluor) (Mw= 70,000 Da), Alexa Fluor 647 N-hydroxysuccinimide ester (A647-NFIS ester), Lysotracker Green and nuclei isolation kit were purchased from Thermo Fisher Scientific (Waltham, USA). Lysosome isolation kit, benzyloxycarbonyl-phenylalanyl-alanyl- fluoromethyl ketone (Z-FA-FMK), human cathepsin B ELISA kit and murine cathepsin B ELISA kit were purchased from Abeam (Cambridge, UK). Anti-nuclear pore complex proteins antibody (anti-NPC) (clone 414) was purchased from Millipore (Burlington, USA). poly(ethylene glycol)-poly{N-[N’-(2-amino-ethyl)-2-aminoethyl]aspartamide}(PEG-pAsp(DET)) was synthesized via the reported method (ChemMedChem., 2006, 1, 439). The molecular weight of the PEG used in this study was 12, 000 Da. The degree of polymerization of pAsp(DET) block in the polymers was determined by 'H-NMR to be 50.1.2. Cell linesMurine colorectal adenocarcinoma CT26 cells, murine triple negative breast cancer 4T1 cells, human pancreatic adenocarcinoma BxPC3 cells, human colorectal adenocarcinoma HT29 cells, human colorectal carcinoma HCT116 cells, murine melanoma Bl 6F 10 cells, murine macrophage RAW264.7 cells, human embryonic kidney HEK293 cells and luciferase-expressing human cervical carcinoma Hela-Luc cells were obtained from Riken BioResource Center (Tsukuba, Japan). For the Luciferaseexpressing murine pancreatic ductal adenocarcinoma KPC (KPC-Luc) cells, KPC cell line was first obtained from Dr. Ryo Tsumura at National Cancer Center in Kashiwa, Japan from a spontaneous pancreatic tumor in a transgenic LSL-KrasG12D / +, LSL- Trp53R172H / +, and Ptfla-Cre mouse (Cancer Sci., 2019, 110, 3296). Next, the cells were transduced with a recombinant retroviral vector encoding a neomycin-resistance gene and Luc-2 gene to get the KPC-Luc cells.CT26, KPC-Luc, B16F10, RAW264.7, HEK293 and Hela-Luc cells were cultured in DMEM completed with 10% FBS and 1 xpenicillin-streptomycin under 37 °C and 5% CO2 atmosphere. 4T1, BxPC3, HT29 and HCT116 cells were cultured in RMPI completed with 10% FBS and 1 xpenicillin-streptomycin under 37 °C and 5% CO2 atmosphere.1.3. Preparation of the probes and controlling formulationsFor constructing the CTSB probe (Peras) and CTSB controlling formulation (CCTSB), BSA (10 mg / mL) was dissolved in 0. IM bicarbonate sodium buffer (pH 8.5). The CTSB- sensitive linker, Azido-PEGs-Val-Cit-PAB-PNP, was dissolved in dimethylformamide (DMF) to a concentration of 10 mg / mL. Fifty times BSA-equivalent Azido-PFGa-Val- Cit-PAB-PNP was mixed into BSA solution, followed by 12 h incubation under 4 °C with continuous shaking. The mixture was then filtered through centrifugation filter tubes (MWCO = 10,000 Da) to remove the unreacted Azido-PEGa-Val-Cit-PAB-PNP. The purified product was then re-dissolved in PBS (pH 7.4) to a concentration of 1 mg / mL on BSA basis. Next, fifty times BSA-equivalent Cy5-DBCO or five times BSA-equivalent A488-DBCO were added into the BSA solution for synthesizing Peras and CCTSB, respectively. After 12 h incubation under 4 °C, the products were purified by filtration through Sephadex G-25 gel to remove the unreacted Cy5-DBCO or A488-DBCO dyes.For constructing the protein degradation probe (Pdeg) and controlling formulation (Cdeg), BSA (10 mg / mL) was dissolved in 0. IM bicarbonate sodium buffer (pH 8.5). Fifty times BSA-equivalent Cy5 NHS ester or five times BSA-equivalent A488 NHS ester were added into the BSA solution for synthesizing Pdeg and Cdeg. After 12 h incubation under4 °C, the products were purified by filtration through Sephadex G-25 gel to remove the unreacted dyes.For determining the degree of labeling (DOL) (average numbers of fluorescent dye molecules conjugated to one BSA molecule), the probes or controlling formulations were dissolved in pure water. Concentrations of the fluorescence dye were measured by UV- Vis absorbance at the maximal absorbance wavelength of corresponding dyes (488 nm for A488 and 650 for Cy5), and BSA concentrations were measured by BCA assay. The DOL of the probes and the controlled formulations were calculated by the ratio of dye concentration to BSA concentration and summarized in Table 1.Table 1. Degree of labeling (DOL) of the probes and controlling formulationsFormulation DOLPeres 6P deg 6CCTSB 3Cdeg 31 .4. Characterization of the probes and controlling formulationsThe emission spectra of the probes and controlling formulations were confirmed firstly.For Peres and Ceres, they were dissolved in PBS (PH 6.5) to a concentration of 5 uM on BSA basis. To confirm the activation of Peres by CTSB enzyme, 5pM CTSB was supplemented to the Peres or Ceres solution and the mixture were incubated under 37°C for 24 h. To normalize the fluorescence signals, free Cy5-NHS ester or A488-NHS ester samples were prepared by dissolving the dyes in PBS (PH 6.5) to the same dye equivalent concentration with the Peres and Ceres solutions respectively. The emission spectra of these samples were measured at corresponding excitation wavelength of the dyes (650 nm for Cy5 and 488 run for A488) by a multi-well plate reader (Spark, Tecan).For Pseg and Cdeg, they were dissolved in PBS (PH 7.4) to a concentration of 5 LIM on BSA basis. To confirm the activation of Psegby protease, 5pM trypsin was supplemented to the Pseg and Cdeg solution and the mixture were incubated under 37°C for 24 h. To normalize the fluorescence signals, free Cy5-NHS ester or A488-NHS ester samples were prepared by dissolving the dyes in PBS (PH 7.4) to the same dye equivalent concentration with the Peres and CCTSB solutions respectively. The emission spectra of these samples were measured at corresponding excitation wavelength of the dyes (650 nm for Cy5 and 488 nm for A488) by a multi-well plate reader.To determine the activation rate of the probes after treatment with lysosomal enzymes, lysosomes were isolated and lysed from CT26 cells by lysosome isolation kit under theinstructions provided by manufacturer. However, for retaining the bioactivity of the protease, protease inhibitors cocktail was excluded during lysosome isolation operation. The CTSB concentration in the lysosome lysate was determined by ELISA kit and the lysate sample was then diluted in PBS (pH 6.5) to make the CTSB concentration be 1 pM. To selectively block the bioactivity of CTSB in the lysate sample, 5 pM CTSB inhibitor Z-FA-FMK was supplemented to the lysosome lysate. To block the bioactivity of general protease in the lysate sample, protease inhibitors cocktail (with concentration suggested by the manufacturer) was supplemented to the lysosome lysate. PCTSB or P^g (5 pM) were dissolved in the prepared lysosome lysate samples (non-treated, Z-FA-FMK treated, or protease inhibitors cocktail treated) separately and incubated under 37°C. At determined time points (1-, 4-, 8-, 16-, and 24-h), fluorescence intensities of the mixtures were measured by a fluorescence spectrometer (NanoDrop 3000, Thermo Fisher Scientific). Fluorescence intensities were normalized by the standard intensity from equivalent free Cy5 dye.To determine the selectivity of PCTSB towards different enzymes, 5 pM PCTSB was incubated in PBS (pH 6.5) containing different enzymes (CTSB, CTSL, MMP-1 or MMP- 3; 0.1 pM final concentration) under 37°C. At determined time points (4-, 8- and 24-h), fluorescence intensities of the samples were measured by fluorescence spectrometer, and the intensities were normalized by the standard intensity from equivalent free Cy5 dye.To determine the activation of the probes under different CTSB concentration, Probes (5 pM) were incubated in PBS (pH = 6.5) containing different CTSB concentration under 37°C. At determined time points (1-, 4-, 8-, 16-, and 24-h), the fluorescence intensities of the mixtures were measured by a fluorescence spectrometer and normalized by the standard intensity from equivalent free Cy5 dye to indicate the activation of the probes.1.5. Detection of endo / lysosomal CTSB activity level and protein degradation rate in cells by the probesCT26, 4T1, KPC-Luc, BxPC3, HT29, HCT116, B16F10, RAW264.7, HEK293 and Hela-Luc cells were seeded to 8-well chambered plates (104cells / well) and cultured under proper condition described in Cell lines. For detecting the activity of endo / lysosomal CTSB, 1 pM PCTSB and 1 pM CCTSB were added in cell culture medium. For detecting the protein degradation rate, 1 pM Pdeg and 1 pM Cdeg were added in cell culture medium. To confirm the activation of the probes in endo / lysosomes, the cells were incubated with 1 pM PCTSB or Pdeg and the endo / lysosomes were stained by Lysotracker Green. For preparing negative control samples with the endo / lysosomal CTSB activity blocked, cells were incubated in medium containing 10 pM Z-FA-FMK for 12 h, then change to incubate in medium containing probes and controlling formulations. After incubation with the probes and controlling formulations for determined time points (1-, 4-, 8-, 16-, 24- and 48-h), cells were washed by PBS containing 2% FBS, and imaged by confocal laser-scanning confocal microscopy (CLSM) (LSM-780, Zeiss) with 488 nm and 633 nm excitation lasers (emission filters: 510 nm and 660 nm, respectively). The co-localizationcoefficients of red channel (probes) to green channel (controlling formulations) were calculated by ZEN software. For quantifying the CTSB activity and protein degradation rate, images of the cells were analyzed by Image J software following the workflow shown in Figure 10. Areas with intensive red pixels were selected via “Moments” autothresholding and recognized as endo / lysosomes and other areas were excluded to filter the green signals (signals of controlling formulations) from cell-membrane and extracellular spaces. The intensities of red channel and green channel in single endo / lysosomes were measured, and signal ratio (red to green) was calculated. The mean red to green fluorescence signal ratio in the endo / lysosomes after 24 h incubation in each kind of cell was defined as the termination point where all the probes were activated. The mean red to green fluorescence signal ratio values of other time points were normalized by the value at 24 h to indicate the activation degree of the probes at determined time.1.6. Investigation of the activation site of probes in endo / lysosome systemCT26 and Hela-Euc cells were seeded and cultured in 8-well chambered plates (104cells / well). The cells were then transfected with CellLight Early Endosomes-GFP, CellEight Late Endosomes-GFP or CellLight Lysosomes-GFP kits accordingly to label the early endosomes, late endosomes and lysosomes with GFP, respectively. The labelled cells were incubated in medium containing 1 pM A647-BSA, PCTXB, or Pc / egfor determined time (4-, 8- and 16-h), then washed by PBS containing 2% FBS. The cells were treated with Hoechst 33342 to stain the nuclei, then observed by CLSM with 405 nm, 488 nm and 633 nm excitation lasers (emission filters: 460 nm, 510 nm and 660 nm, respectively). To quantify the localization of A647-BSA or probes in different endo / lysosome compartments, the fraction of red fluorescence localizing at the corresponding compartments was analyzed by Zen software using the following equation:Signal of red pixels localized at green pixels Localization fraction = - - - - — - - — - - - -Overall siganl of red fluoresence pixels1.7. Quantification of CTSB abundance in different cell linesCT26, 4T1, KPC-Luc, BxPC3, HepG2, HT29, HCT116, B16F10, RAW264.7, HEK293 and Hela-Luc cells were collected for preparing whole-cell (WC) lysate or extracted endo / lysosome (Els) lysate. For collecting the WC lysate, the cells were treated by RIPA lysis buffer supplemented with protease inhibitor cocktail. For collecting Els lysate, endo / lysosome were isolated via the reported method[4]. Total protein concentration in these samples was quantified by BCA assay, and samples were then diluted in PBS for getting the same final protein concentration. CTSB concentrations in the samples were then quantified by ELISA kits and converted to relative values for correlation analysis between CTSB abundance and the activation degree of the probes at 4h by GraphPad Prism software.1.8. Determine the pH-dependence of CTSB activity.Pers® (5 pM) was incubated with CTSB (0.1 pM) in PBS with different pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.4). The activation rate of PCTSB was monitored by measuring the fluorescence intensity of the solution at different time points. The intensities were normalized to the standard intensity from equivalent free Cy5 dye, and the normalized intensity of each solution after 4 h incubation was plotted to indicate the relative CTSB activity under corresponding pH.1.9. Quantification of acidification rate of endo / lysosomes in different cell lines.The quantification of endo / lysosome pH in cell lines were conducted following reported protocol^. CT26, 4T1, KPC-Luc, BxPC3, HT29, HCT116, B16F10, RAW264.7, HEK293 and Hela-Luc cells were seeded and cultured in 8-well chambered plates (104cells / well). TMR-Dextran-Fluor (50 pg / mL) was then added to cells. After 4 h incubation, cells were washed by PBS containing 2% FBS and imaged by CTSM with 488 nm and 543 nm excitation lasers (emission filters: 510 nm and 570 nm, respectively). The images from the two channels were analyzed by Image J software following the workflow shown in Figure 16. Areas with intensive red pixels were selected via “Moments” autothresholding and recognized as endo / lysosomes and other areas were excluded to eliminate the extracellular signals. The intensities of red channel and green channel in single endo / lysosomes were measured, and signal ratio (red to green) was calculated. To determine the definite value of pH, calibration curves were obtained from corresponding cells. The cells were cultured with TMR-Dextran-Fluor (50 pg / mL) for 4 h, followed by washing with 2% FBS -contained PBS and then treated with 20 pM nigericin and 20 pM monensin in buffers (100 mM phosphate and 150 mM NaCl) with different pH value respectively for 5 min, then imaged by CLSM. The fluorescence intensity ratio was then converted to pH values according to the calibration curve and plotted to indicate the pH distribution of the endo / lysosomes in different cells.1.10. Preparation and characterization of antibody-polymer conjugateIn this research, anti-nuclear pore complex antibody (anti-NPC) was used as the model antibody. The anti-NPC antibodies were firstly labeled by Cy5 NHS ester. The anti-NPC antibodies were dissolved in 0.1M bicarbonate sodium buffer (pH 8.5), and BSA-equivalent Cy5 NHS ester was added into the BSA solution. After 12 h incubation under 4 °C, the antibodies were purified by filtration through Sephadex G-25 gel to remove the unreacted dyes.For conjugating the CTSB-sensitive linker to the antibodies, anti-NPC was dissolved in 0.1M bicarbonate sodium buffer (pH 8.5) to a concentration of 10 mg / mL. Fifty times anti-NPC-equivalent Azido-PEGa-Val-Cit-PAB-PNP was added into the anti-NPC solution, followed by 12 h incubation under 4 °C with continuous shaking. The mixture was then filtered through centrifugation filter tubes (MWCO = 10,000 Da) to remove the unreacted Azido-PEGs-Val-Cit-PAB-PNP and get the modified antibody (anti-NPC / 1). To determine the number of Azido-PEGa-Val-Cit-PAB-PNP conjugated to one antibody, theanti-NPC / 1 was prepared by non-labeled anti-NPC and dissolved in PBS (pH 7.4) to a concentration of 10 mg / mL on anti-NPC basis, then fifty times anti-NPC-equivalent Cy5- DBCO was added to the anti-NPC / 1 solution. The mixture was kept reacting for 12 h under 4 °C, then filtered through Sephadex G-25 gel to remove the unreacted Cy5-DBCO. The antibody concentration in the purified product was determined by BCA assay, and the Cy5 concentration was determined by the absorbance at 650 nm. The number of Azido-PEGi-Val-Cit-PAB-PNP conjugated to one antibody was calculated to be 10.For conjugating the polymer to anti-NPC / 1, PEG-pAsp(DET) was dissolved in 0.1M bicarbonate sodium buffer (pH 8.5), to a concentration of 10 mg / mL and reacted with equivalent DBCO-PEG4-NHS ester for 12 h under 4 °C. The product was purified by dialysis against water (membrane MWCO: 6, 000-8, 000 Da) to remove the unreacted DBCO-PEG4-NHS ester. Next, anti-NPC / 1 was dissolved in PBS (pH 7.4) to a concentration of Img / mL and reacted with fifty times anti-NPC-equivalent DBCO- conjugated PEG-pAsp(DET) for 12 h under 4 °C. The polymer-conjugated anti-NPC antibodies (anti-NPC / l / p) was purified by centrifugal filtration (MWCO: 50, 000 Da).For characterize the size of anti-NPC / l / p, the anti-NPC / l / p or free anti-NPC sample were measured by fluorescence correlation spectroscopy equipped in CLSM with 633 nm excitation laser and 660 nm emission filter. The diffusion time values of the samples were recorded. For calculating the diffusion coefficients of anti-NPC / 1 and free anti-NPC, free Cy5 NHS ester was also measured by FCS as a standard reference. The diffusion coefficient of the sample was calculated by the following equation.Dcy5 and Tcys are the diffusion coefficient and diffusion time of free Cy5 NHS ester, respectively. Dsampie and Tsampie are the diffusion coefficient and diffusion time of sample (anti-NPC / l / p or free anti-NPC antibody), respectively. Dcys was reported to be 280 pm2S'1 [6]. The hydrodynamic diameters of anti-NPC / l / p or free anti-NPC antibody were calculated from the obtained diffusion coefficients by Stokes-Einstein equation, as follows.d is the hydrodynamic diameter of the sample, kp is Boltzmann’s constant. T is the temperature during the measurement. r| is the dynamic viscosity of the sample. D is the diffusion coefficient of the sample obtained from FCS measurement.The molecular weight distribution of the anti-NPC / l / p was analyzed by HPLC measurement (LC-Extrema, JEOL) (Column: Superdex 200-10 / 300GL, eluent: 10 mM PBS pH 7.4; temperature: 25 °C; flow rate: 0.75 mL min-1; detector: UV 280 nm). To determine the dissociation of the anti-NPC / l / p upon CTSB incubation, anti-NPC / l / p (5 |1M) was incubated with 0.1 pM CTSB in PBS (pH 6.5) under 37 °C for 24 h, then analyzed by HPLC.To determine the dependence of anti-NPC / l / p dissociation on environmental CTSBlevel, anti-NPC / l / p (5 pM) was incubated under different CTSB concentration (0.1 pM, 1 pM and 5 pM). At determined time points (1-, 4-, 8-, 16- and 24-h), the samples were measured by FCS, and the sample diffusion time values were recorded and normalized to the diffusion time of anti-NPC / l / p.1.11. In vitro delivery of antibody by antibody -polymer conjugateCT26, 4T1, KPC-Luc, BxPC3, HT29, HCT116, B16F10, RAW264.7, HEK293 and Hela-Luc cells were cultured in 8-well chambered plates (104cells / well). The cells were incubated in medium containing 1 pM anti-NPC / l / p. In negative control samples, the endo / lysosomal CTSB activity of the cells were blocked, by incubating the cells firstly in medium containing 10 pM Z-FA-FMK for 12 h, and then changed to medium containing 1 pM anti-NPC / l / p. After incubation with anti-NPC / l / p for determined time. The cells were washed by PBS containing 2% FBS. The nuclei of the cells were stained by Hoechst 33342, and the endo / lysosomes were stained by Lysotracker Green. The cells were then observed by CLSM with 405 nm, 488 nm and 633 nm excitation lasers (emission filters: 460 nm, 510 nm and 660 nm, respectively). The co-localization coefficients of red channel (anti-NPC antibodies) to green channel (Lysotracker) were calculated by ZEN software.For quantifying the delivery efficiency of anti-NPC antibodies, cells were cultured in medium containing 1 pM anti-NPC / l / p or 1 pM free anti-NPC antibodies. After 24 h incubation, the cells were harvested, and the nuclei were isolated from the cells by nuclei isolation kit. The nuclei were then lysed by RIPA lysis buffer supplemented with protease inhibitors cocktail. The protein concentrations of the nuclei lysate samples were quantified by BCA assay, and all the samples were diluted respectively to get the same final protein concentration (5 mg / mL). The fluorescence intensities from the Cy5 in the lysate samples were measured by fluorescence spectrometer (NanoDrop 3000, Thermo Fisher Scientific). The ratio of the intensity from anti-NPC / l / p treated sample and the intensity from free anti-NPC antibody treated sample was defined as the normalized fluorescence intensity, indicating the ability of anti-NPC / l / p to improve the delivery efficiency of the antibodies to target antigen.1.12. Statistical analysisAll statistical analysis in this study was performed by GraphPad Prism (v8.4.3). For bar graphs, the p values were calculated by One-Way ANOVA. For correlation analysis, Pearson r values and two-tailed p values were calculated for evaluating the correlation between every two parameters.2. Results and discussionThe fluorescence emission spectra of the probes confirmed the existence of the self- quenching effect (Figure 1 A). Compared with equivalent free Cy5 molecules, both PCTSBand Pdeg showed diminished fluorescence. After incubation with the corresponding enzymes, the fluorescence emission of the probes increased, indicating that the quenching effect between the Cy5 dyes was relieved. On the other hand, both of the “always-on” controlling formulations (CCTSB and C*g) showed constant fluorescence. To confirm the activation of the probes by lysosomal enzymes, the lysosomes were isolated from CT26 cells and lysed to get a mixture of lysosomal enzymes. Both PCTSB and Pdeg showed activation after incubation with the lysosomal enzyme mixture, confirmed by the gradual increase in the fluorescence emission (Figure IB). By adding the CTSB inhibitor Z-FA- FMK, the activation of PCTSB was clearly suppressed, indicating that the activation of PCTSB can be preferentially attributed to CTSB rather than to other proteases. This selectivity of Pcrss towards CTSB was further confirmed by incubating PCTXB with purified CTSB and other proteases (Figure 8C).. Thus, PCTSB could be a viable indicator to sense environmental CTSB activity (Figure 8B). In the case of Pc / eg, a protease inhibitor cocktail efficiently reduced its activation (Figure IB), whereas Z-FA-FMK showed slight suppression. Also, incubation with pure CTSB was not effectively activate the Pdeg (Figure 8 C). These results demonstrated that the activation of Pdeg was induced by various lysosomal proteases.The probes showed capability for monitoring the CTSB activity and BSA degradation in cells. After incubating the probes with 4T1 cells, we assess the intracellular fluorescence by confocal laser scanning microscopy (CLSM). Clear red fluorescence was detected from vesicular structures in the intracellular space, which corresponds to activated probes in the endo / lysosomes (Figure 1C). Notably, while the controlling formulations (green fluorescence) showed absorption on the cell surface, the red fluorescence from the probes was only detected intracellularly. Such distribution of the fluorescence signals supports the activation of the probes by intracellular enzymes rather than extracellular environment factors. Also, Z-FA-FMK-treated cells showed diminished PCTSB activation, but barely affected Pdeg activation, supporting the in vitro specificity of PCTSB (Figure 8 D and E).The PCTSB was then applied to track the endo / lysosomal CTSB activity and the Pdeg was used to monitor the protein degradation rate in endo / lysosomes. The probes and the corresponding controlling formulations were incubated together with the cells. The fluorescence ratio of the red channel (PCTSB or Pdeg) to the green channel (CCTSB or Cdeg) in individual endo / lysosome was calculated for indicating the activation degree of the probes (Figure 2 A and D and Figure 10). The average fluorescence ratio (Fcy5 / FA488) in the endo / lysosomes showed a time-dependent increase (Figure 2 B and E). For both PCTSB and Pdeg, the increase of average FCys / FA488 between 24 h and 48 h incubation was modest in CT26 as well as Hela-Luc cells, indicating nearly complete activation of these probes after 24 h incubation. Thus, we set the average FCys / FA488 value at 24 h incubation as the standard reference for normalizing the Fcy5 / FA488 values at other time points to quantify the activation degree of the probes, as defined by the following equation:where A(probe-)(T') is the activated fraction of the determined probe (PCTSB or Pdeg) at a specific time T . FCy5 / FA488(T) represents the average ratio of the fluorescence intensities from Cy5 and A488 in individual endo / lysosomes calculated from the CLSM images at time T. Thus, thecan be used to evaluate the endo / lysosomal CTSB activity, and can indicate the degradation fraction of the BSA proteins.The was measured in different cell lines at defined timepoints (Figure 10). The PCTSB activation rate was found to be variable among cell types (Figure 2C). For example, in CT26 cells, the A(pCTSBy reached around 80% after 8 h incubation, while in Hela-Luc cells this value was approximately 30%. Moreover, the differentiation of fl(;pCTSB) occurred during an early period (t < 8 h). After 16 h incubation, all the cells showed high A(pCTSB) ( >70%). On the other hand, the activation rate of Pdeg was highly synchronous among all the cell lines (Figure 2 F). In all the cells, Pdeg showed low activation at earlier time point (^(pde5) (4 h) < 30 %), and upon longer incubation, high activation fraction of Pdeg was found in all the cells (16 h) >50%). Thus, we concluded that the endo / lysosomal CTSB activity level described by A^PCTSB^ was highly differentiated in some cancer cells during the earlier stages of endocytosis.We then tried to distinguish the contributions of the different compartments in the endo / lysosome system to the activation of the probes. Early endosomes (EEs), late endosomes (LEs) and lysosomes were marked with the corresponding GFP-tagged proteins to separately visualize the sub-components of the endo / lysosome system with green fluorescence (Figure 11 A). We first confirmed the distribution of the internalized BSA was time dependent. The BSA molecules were mainly found in the EEs at the beginning, and gradually transported to LEs and lysosomes upon longer incubation (Figure 11B), consistent with the endocytosis pathway. In CT26 cells, Peras showed high activation from early time point and the signal of activated Peras was highly co-localized with the EEs (Figure 11C). On the other hand, in Hela-Luc cells, Peras showed low activation at the early time point, and a longer incubation was needed for the probes to be activated in the lysosomes. These results suggest that the activation site of Peras was different in these cells. In the CT26 cells with higher endo / lysosomal CTSB activity, PCTSB could be activated in EEs, while in cells with lower endo / lysosomal CTSB activity like Hela-Luc cells, Peras needed to reach the lysosomes for getting activated. On the other hand, the activation site for P^gwas mostly in the lysosomes for both cell lines (Figure 11D). Thus, the difference of CTSB activity level among the CT26 and HeLa-Luc cellscould be mainly atributed to the differences in their EEs. This observation may also explain the results in Figure 2, where the differential Peras activation rate was only detected at early incubation time, since longer incubation will lead the probes into the lysosomes, where they will be degraded by proteases.The mechanism behind this differentiated endo / lysosomal CTSB activity was further explored. As previous studies have confirmed CTSB expression is upregulated from the transcription level in cancer cells

[0029] , we firstly determined the abundance of CTSB hi cells by measuring CTSB concentration in lysate of whole cells (WC) or isolated endo / lysosomes (ELs) via ELISA method. In both fractions, we observed a variation of the CTSB concentration among the cell lines (Figure 3A). Then, we analyzed the correlation between the CTSB abundance and the endo / lysosomal CTSB activity (represented as A(pCTSH)at 4 h measured in Figure 2C). Surprisingly, the CTSB concentration in in ELs did not correlate with the activation of the Peras lysates (Pearson r coefficient = 0.01) (Figure 3B). Moreover, the activation of the Peras was only moderately correlated with the total CTSB in WC (Pearson r coefficient = 0.49). These results suggest that the differential endo / lysosomal CTSB activity may not be simply atributed to the abundance of CTSB. Considering that the enzymatic activity of CTSB is pH-dependent and has the optimum between pH 4.5 and pH 5.5|301, we hypothesized that the activity of the CTSB in the cells was different due to alterations in endo / lysosomal acidification. In fact, recent studies have revealed that cancer cells have dysregulated endo / lysosomal acidification131,32], and the endo / lysosomal acidification rate was observed to be variable among cells types[20’34]. Thus, we used a commercial pH-sensitive probe, tetramethylrhodamine and fluorescein labeled dextran (TMR-Dextran-Fluor) to evaluate the acidification of endo / lysosomal system. As the emission intensity of fluorescein is pH-responsive, the fluorescence ratio of fluorescein (green channel) to TMR (red channel) can be used for indicating the pH of endo / lysosomal system (Figure 12 and Figure 13). Our result indicated that the acidification rate of the endo / lysosomes varied among cell lines (Figure 3C). For example, the average endo / lysosomal pH of CT26 cells after 4 h incubation was around 5.2, while in Hela-Luc cells this average pH was 5.9 (Figure 3D). As the optimal pH (pHopt) for cleaving the VaLCit linker by CTSB was determined to be 5.0 (Figure 14), we defined a coefficient, i.e., the optimal pH convergence index (OPCI), to evaluate the convergence of the pH distribution of the endo / lysosome system toward the pHopt, as follows:where the pHi are the pH values of individual endo / lysosomes and pHopZis 5.0. A clear variation of OPCI among cell lines was found (Figure 3D). In the cell lines having higher endo / lysosomal CTSB activity, acidified endo / lysosomes with pH close to pl !0 / p were detected. The Peras showed a strong correlation with the OPCI (Pearson r coefficient = -0.85) (Figure 3E), indicating that the optimal endo / lysosomal microenvironment is more significant for CTSB activity than just the abundance of the CTSB protein.Based on the above findings, we designed a CTSB-sensitive system for intracellular delivery of proteins. The anti-nuclear pore complex antibody (anti-NPC) was used as the model protein. The endosomolytic polymer, poly (ethylene glycol)-poly{N-[N’-(2-amino- ethyl)-2-aminoethyl]aspartamide} (PEG-pAsp(DET)) was conjugated to the anti-NPC antibodies via the CTSB-sensitive Val-Cit linker to form the antibody-loaded nanocarrier (anti-NPC / l / p) (Figure 7). The flanking 1 ,2-diaminoethane moieties presented in the PEG-pAsp(DET) polymer have been confirmed to have strong membrane destabilization effect under endo / lysosomal acidic pH1351. Thus, we hypothesized that the anti-NPC / l / p could sense the endo / lysosomal CTSB activity among cell-lines to release the endosomolytic polymers in the endo / lysosomes at different rate. Thus, the fast release of PEG-pAsp(DET) from the anti-NPC / l / p caused by high endo / lysosomal CTSB activity in some cells may lead to effective endosomal escape, allowing the antibody to enter the cytosol and finally recognize the antigen on the cell nucleus. However, in cells with lower endo / lysosomal CTSB activity, the ineffective release of the PEG-pAsp(DET) could not aid the antibody escape from endo / lysosomes, which would lead to lysosomal compartments where the antibodies are degraded.Fluorescence correlation spectroscopy (FCS) confirmed the increased molecule size of anti-NPC / l / p compared to native anti-NPC, indicating the successful polymer conjugation (Figure 4 A and B). The anti-NPC / l / p dissociated with CTSB, and the dissociation rate was sensitive to the CTSB level (Figure 4 C and D). To test the CTSB- differentiated delivery efficiency of the antibody, we studied the endosomal escape and intracellular targeting of anti-NPC / l / p in CT26 cells with high CTSB activity and Hela- Luc cells with low CTSB activity. Contrasting endosomal escape was found in the two cells, indicated by the co-localization coefficient of the Cy5-labeled anti-NPC antibodies with endo / lysosomes (Figure 5A). Moreover, the antigen-recognition of the anti-NPC antibodies in CT26 cells was confirmed from the CLSM images. To quantitatively evaluate the antibody delivery efficiency, we extracted the nuclei from the cells incubated with anti-NPC / l / p for 24 h and detected the fluorescence intensity from these nuclei. As a reference, we also extracted the nuclei from the cells incubated with same amount of free anti-NPC antibodies. The fluorescence signal from anti-NPC / l / p treated nuclei was normalized to the signal from equivalent nuclei of cells treated with free anti-NPC antibodies. The normalized fluorescence intensity clearly confirmed the high targeting ability of anti-NPC / l / p in CT26 cells compared to Hela-Luc cells (Figure 5B). Moreover, by treating the CT26 cells with a CTSB inhibitor, the delivery efficiency of anti-NPC / l / p was reduced, confirming the performance of this delivery system was dependent to the endo / lysosomal CTSB activity. Next, we screened the delivery efficiency of anti-NPC / l / p in several cell lines (Figure 15 A). We quantified the delivery by the normalizedfluorescence intensity from the extracted nuclei (Figure 5C). For evaluating the correlation between the endo / lysosomal CTSB activity and the antibody delivery efficiency, we defined a coefficient, i.e. , the effective CTSB activity (EACTXB), which is calculated as follows:whererepresent the activation fraction of PCTSB andPdeg at time T described in Equation (1). Thus, the EACTSB would represent the quantity of the anti-NPC / l / p activated by the CTSB while subtracting the fraction of degraded antibodies. EACTSB varied in the cell lines (Figure 15B). The delivery efficiency of anti- NPC / l / p strongly correlated with the EACTSB (Figure 5D and Figure 15C and D), supporting the effect of CTSB activity on the intracellular delivery of the antibodies.In conclusion, we found a differentiated endo / lysosomal CTSB activity in cancer cells and exploited it to achieve selective endosomal escape with CTSB-sensitive nanocarriers. Our approach allowed specific delivery of antibodies with high selectivity towards cells with high CTSB activity. 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Claims

CLAIMS1. A cathepsin B-sensitive probe comprising a conjugate of a cathepsin B- sensitive linker and a fluorescent emitter.

2. The probe according to claim 1, wherein the cathepsin B-sensitive linker comprises a valine-citrulline peptide.

3. The probe according to claim 2, wherein the linker containing the valinecitrulline peptide is azido- [poly (ethylene glycol)]3-valyl-citrullyl-(4-aminobenzyl)-(4- nitrophenyl) carbonate.

4. Protease-sensitive probe comprising a conjugate of protease-sensitive protein and a fluorescent emitter.

5. A protein-probe complex comprising the probe according to claim 1 bound to a protein.

6. The complex according to claim 5, wherein the protein is bovine serum albumin.

7. A method for detecting a cell having a cathepsin B activity, comprising contacting the probe according to any one of claims 1 to 3 or the complex according to claim 5 or 6 with a test cell.

8. The method according to claim 7, wherein the cell having a cathepsin B activity is a cancer cell.

9. A method for detecting protease-rich cell, comprising contacting the probe according to claim 4 with a test cell.

10. An intracellular protein delivery system, comprising a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein.

11. A protein delivery kit into a cell, comprising a complex in which a cationic block copolymer is bound to a conjugate of a cathepsin B-sensitive linker and a protein.

12. The system according to claim 10, wherein the protein is an antibody.

13. The kit according to claim 11, wherein the protein is an antibody.

Images