Monomer reactivity screening via model ATRPOne-pot iterative synthesis generally comprises sequential reactions that occur in a single reactor in a preset order without purification of intermediates, providing a high-efficiency platform for fabrication of sequence-controlled polymers. First, the key to one-pot iterative synthesis lies in quantitative monomer conversion during each polymerization cycle. Second, to prevent protein denaturation and maintain the structural intact of proteins during the polymerization process, one-pot iterative living polymerization has to be conducted under mild conditions, i.e., in aqueous medium and at low temperature.To screen highly reactive monomers that can be efficiently polymerized via ATRP under mild conditions, a readily obtained brominated poly(ethylene glycol) (2 kDa) (PEG2k-Br) was chosen as the model macroinitiator to initiate the typical ATRP of a variety of methacrylate- and acrylate-based monomers in an aqueous solution at 35 °C, which allowed real-time monitoring of the polymerization progress by nuclear magnetic resonance (NMR) techniques. The polymerization of methacrylate-based monomers (Supplementary Fig. 21) and acrylamides (N,N-dimethyl acrylamide and N-isopropyl acrylamide) proceeded sluggishly (Supplementary Fig. 22), and never went to completion even after 7 days due to their relatively low rates of propagation (kp) along with high rates of termination51. In contrast, acrylate-based monomers, in particular, the aforementioned MA (M′), OEGA (O′) and HEA (H′), showed quantitative monomer conversion (>99%) as confirmed by 1H NMR spectroscopy with DMF as an internal standard (Fig. 3a and Supplementary Fig. 23), to give well-defined PEG-based diblock copolymers. Therefore, the three reactive acrylates were adopted to produce sequence-controlled protein-multiblock polymer conjugates. In the resulting bioconjugates, the hydrophobic MA block would introduce the hydrophobic interaction with the nonpolar residues on the protein surface, the functional HEA block would enhance the hydrogen-bonding interaction with the polar residues on the protein surface, and the hydrophilic OEGA block could improve the biostability of the protein in vitro or in vivo.Fig. 3: Structural characterization of sequence-controlled BSA-multiblock polymer conjugates.a In situ monitoring of ATRP progress of three acrylates (MA, M′; OEGA, O′; HEA, H′) initiated with PEG2K-Br using 1H NMR spectrometry. The quantitative conversion was confirmed with the disappearance of the vinyl signals at 5.8–6.4 ppm. b, c SEC traces of all growing BSA-multiblock polymer conjugates via QOILP, including (b) all bioconjugate intermediates towards BSA-HHHHHH, and (c) all bioconjugate intermediates towards BSA-MOHMOH. d SEC traces of BSA, BSA-Macro, and six target sequence-controlled BSA-multiblock polymer conjugates. e SDS-PAGE of BSA, BSA-Macro, and six target sequence-controlled BSA-multiblock polymer conjugates. Representative images from three independent experiments are shown. f CD spectra of BSA and sequence-controlled BSA-multiblock polymer conjugates. g Biochemical stability assay of BSA and BSA-multiblock polymer conjugates after heating to 90  °C for 30 min by HPLC (n = 3). h Esterase-like activities of BSA and BSA-multiblock polymer conjugates. The data are shown as mean ± SD (n = 3) for g and h. Source data are provided as a Source Data file.Controlled fabrication of bioactive BSA-multiblock polymer conjugatesThe BSA-macroinitiator (BSA-Macro) was site-selectively synthesized via a biorthogonal Michael addition of maleimide-functionalized ATRP initiator to non-bridged cysteine 34 (Cys34) of BSA under milder conditions (in aqueous solution at 7  oC)55,56, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS, size exclusion chromatography (SEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed to verify the identity of target BSA-Macro (Fig. 3e, Supplementary Figs. 24 and 38). The monomer conversion ratio of three acrylates (MA, OEGA and HEA) via ATRP initiated with BSA-Macro was further analyzed in time by 1H NMR spectroscopy with DMF as an internal standard, and the proton signals at 5.8–6.4 ppm of vinyl groups of all three acrylates finally become undetectable after a certain period of time, demonstrating parallelly quantitative monomer conversion (>99%) (Supplementary Fig. 25).Then, the freshly prepared BSA-Macro as a macroinitiator was used to initiate the polymerization of MA, OEGA, and HEA, respectively, to produce the BSA-multiblock homopolymer conjugates. SEC was subsequently utilized to examine all growing BSA-multiblock homopolymer conjugates during each polymerization cycle, giving single SEC peaks and further verifying their readily controlled polymerization nature. Along with their chain growth, the SEC retention time of these growing BSA-multiblock homopolymer conjugates gradually shortened, demonstrating a stepwise increase in size and molecular weight (Fig. 3b and Supplementary Fig. 26). After six iterations, three target BSA-multiblock homopolymer conjugates were successfully acquired, including BSA-MMMMMM, BSA-OOOOOO, and BSA-HHHHHH (Supplementary Fig. 27). The structure and molecular weight of the above three bioconjugates were confirmed by 1H NMR spectroscopy (Supplementary Figs. 29–31), SEC (Fig. 3d and Supplementary Fig. 37), SDS-PAGE (Fig. 3e and Supplementary Fig. 38), plus MALDI–TOF–MS (Supplementary Fig. 28 and Supplementary Fig. 35). To verify the controllability of this QOILP strategy, we directly introduced 120 equivalents of acrylates individually at a time in one-pot, and the resultant BSA-polymer conjugates (BSA-120M′, BSA-120O′ and BSA-120H′) exhibited analogous SEC patern, molecular weight and its distribution to those of the aforementioned three BSA-multiblock homopolymer conjugates formed by stepwise addition of 20 equivalents of acrylates for 6 times (Supplementary Fig. 36).To create sequence-controlled BSA-multiblock copolymer conjugates via QOILP, all three acrylates (MA, OEGA, and HEA) were added in one-pot in a preset sequence and initiated with BSA-Macro under the same condition as above. With each addition of acrylates, the resulting mixture was left for continued polymerization for a certain period of time, ensuring complete monomer conversion (>99%) during each polymerization cycle. All growing BSA-multiblock copolymer conjugates were analyzed by SEC. Likewise, the SEC retention time of the growing bioconjugate intermediates gradually reduced as the bioconjugates grew in length, verifying a stepwise rise in molecular weight (Fig. 3c and Supplementary Fig. 26). After six iterations, three target sequence-controlled BSA-multiblock copolymer conjugates with defined sequences and specific properties were successfully produced, including BSA-MMOOHH, BSA-MOHMOH, and BSA-OMOHHM, which were further identified by 1H NMR (Supplementary Figs. 29–34), SEC (Fig. 3d and Supplementary Fig. 37), SDS-PAGE (Fig. 3e and Supplementary Fig. 38), and MALDI–TOF–MS (Supplementary Figs. 28 and Supplementary 35). We noted that the appearance of multiple peaks in SEC of all BSA bioconjugates might be attributed to the formation of multimers of BSA (i.e., dimer, trimer, etc.) as well as the resulting clusters of BSA bioconjugates via the interchain entanglement of polymers in solution (Supplementary Fig. 37).To demonstrate the applicability of this QOILP approach, we further examined and evaluated the polymerization activity of two more protein-based macroinitiators, including GOx-Macro and β-gal-Macro. As depicted in Supplementary Figs. 25 and 26, the quantitative monomer conversion (>99%) of three acrylates (MA, OEGA, and HEA) could be achieved using both GOx-Macro and β-gal-Macro as macroinitiator, validated by in time 1H NMR spectroscopy monitoring (Supplementary Figs. 39a–c and 40a–c), the shortened retention time in SEC (Supplementary Figs. 39d and 40d). 1H NMR spectroscopy (Supplementary Figs. 39e–g and 40e–g), FTIR spectroscopy (Supplementary Figs. 39h and 40 h) and SDS-PAGE (Supplementary Figs. 39i and 40i) further proved the successful formation of bioconjugates, and verified the identity of the resultant GOx-, and β-gal-polymer conjugates (i.e., GOx-M50, GOx-O50, GOx-H50, β-gal-M50, β-gal-O50, and β-gal-H50). We noted that, all GOx bioconjugates show similar activity to the native GOx (Supplementary Fig. 39j), while all β-gal bioconjugates exhibited lower activity than that of the native β-gal (Supplementary Fig. 40j). The results showed that the bioconjugation of GOx had a slight effect on the activity of GOx, and the bioconjugation of β-gal reduced the activity of β-gal to a certain degree. It was noting that the physicochemical properties (i.e., isoelectric point, the secondary structure arrangement, and the surface charge) of proteins (i.e., BSA, GOx, and β-gal) had no effect on the synthesis efficiency of this QOILP approach, demonstrating its universality and wide applicability for the creation of a variety of protein-multiblock polymer conjugates.Physicochemical properties of BSA-multiblock polymer conjugatesBSA exhibited characteristic circular dichroism (CD) spectra with two negative brands near 209 nm and 220 nm, and a positive peak around 192 nm due to its α-helix structure. In Fig. 3f, BSA-Macro and all six sequence-controlled BSA-multiblock polymer conjugates showed pretty analogical CD bands compared to that of BSA, in particular, a strong positive peak at 191 nm (π → π*) and a characteristic negative doublet at 209 (π → π*) and 220 nm (n→π*) corresponding to α-helix conformation, indicating that the secondary structure of BSA remained intact during the whole synthesis and polymerization process. Dynamic light scattering (DLS) was carried out to evaluate the hydrodynamic diameters (Dh) of BSA and its bioconjugates in water. The pristine BSA had a hydrodynamic size of ca. 4.46 ± 0.77 nm, while BSA-multiblock polymer conjugates showed increased size in the range of 11.08 ± 0.82 nm to 28.76 ± 1.60 nm (Supplementary Fig. 41 and Supplementary Table 1). The increase in size further confirmed the successful fabrication of BSA-multiblock polymer conjugates. Meanwhile, the sequence alteration of hydrophobic monomer (MA), hydrophilic monomer (OEGA) and hydrogen-bond donor (HEA), had a great effect on the morphologies and aggregated behaviors in solution, eventually leading to a noticeable variation in Dh of the resultant bioconjugates. Both BSA-MMMMMM (17.50 ± 2.91 nm) and BSA-HHHHHH (19.06 ± 0.68 nm) demonstrated relatively larger Dh than that of BSA-OOOOOO (11.08 ± 0.82 nm). All-atom MD simulation verified that BSA-MMMMMM revealed maximum hydrophobic interactions while BSA-HHHHHH had the most intra- or inter-molecular hydrogen bonds compared to BSA-OOOOOO, which further led to the aggregation of BSA-MMMMMM or BSA-HHHHHH in solution to some degree and the subsequent formation of bioconjugate clusters. Compared to BSA-multiblock homopolymer conjugates, the hydrophilicity/hydrophobicity of BSA-multiblock copolymer conjugates could be readily regulated via precisely controlling over the monomer sequence of the grafted copolymer in BSA bioconjugates, in particular, BSA-MMOOHH and BSA-MOHMOH, revealed relatively larger Dh than that of BSA-OMOHHM (Supplementary Table 1).Indeed, tens of proton-donor residues in BSA, exhibited esterase-like activities towards p-nitrophenyl acetate, to hydrolyze p-nitrophenyl acetate to form acetylated adducts and p-nitrophenol57. The esterase-like activity of BSA and BSA-multiblock polymer conjugates were then evaluated by monitoring the hydrolysis of p-nitrophenyl acetate (Fig. 3h and Supplementary Table 2). In Fig. 3h, the resulting BSA bioconjugates had similar yet distinct esterase-like activities towards p-nitrophenyl acetate. The esterase-like activity of these bioconjugates would principally depend on the surrounding environment of the corresponding active residues of BSA (Supplementary Fig. 47). In particular, the conjugated multiblock polymers to BSA might bury the active residues of BSA via hydrogen bonds to inhibit the proton transfer catalysis to different degree. We noted that, all BSA bioconjugates retained more than 80% esterase-like activity of the BSA, noteworthily, BSA-MMMMMM exhibiting nearly 100% esterase activity of BSA, while BSA-HHHHHH having the minimum esterase activity (ca. 83.92 ± 1.93%) of BSA (Supplementary Table 2). All-atom MD simulations demonstrated that BSA-HHHHHH revealed maximum hydrogen bonds in solution (Fig. 4d), which gave rise to the burial of six active catalytic residues of BSA in total (Supplementary Table 3), ultimately leading to a reduction in esterase-like activity. In contrast, BSA-MMMMMM had no hydrogen bonding with BSA (Fig. 4d), in this case, the active catalytic residues of BSA would almost stay undisturbed, further resulting in a much higher catalytic activity retention.Fig. 4: Sequence-dependent protein/polymer interactions elucidated by all-atom MD simulations.a The last-frame MD snapshots of six BSA-multiblock polymer conjugates with different monomer sequences. The multiblock polymer is shown by sticks and colored by monomer type (pale blue for monomer M′ or MNM, pale red for monomer O′ or MNO, and pale green for monomer H′ or MNH). The interaction residues of BSA with the multiblock polymer are represented on the surface and colored according to their hydrophobic nature (blue) or hydrophilic nature (red). The interaction residues are determined using a distance cut-off of 6 Å, and the overall surface representation of BSA is shown on the left. b Buried surface area of BSA in each conjugate system calculated for all residues (left), polar residues (middle), and nonpolar residues (right). c The total interaction energies between BSA and the multiblock polymer for each conjugate system (left), and the contributions of the electrostatic and van der Waals interactions are shown in the middle and right panels. The error bars in b and c show the average values over the last 200-ns MD simulations, with the corresponding standard deviations. d Counts of hydrogen-bonds between BSA and the multiblock polymer. e Counts of hydrophobic contacts between BSA residues and the methyl group of monomer M′. The counts of contacts were calculated only for the heavy atoms, with a distance cut-off of 4.5 Å. f Decomposition of the interaction energy according to each monomeric unit in distinct BSA-multiblock polymer conjugate systems. The data in b–f show average values over 200 frames with an interval of 1 ns from the last 200-ns MD simulations. The error bars in b–e show standard deviations. Source data are provided as a Source Data file.The thermostability of sequence-controlled BSA-multiblock polymer conjugates was examined by SEC using an accelerated heating test37. After being heated up to 90 °C for 30 min, the resultant BSA-multiblock polymer bioconjugates yet held much higher intact BSA content (>90%) compared to the pristine BSA (Fig. 3g, Supplementary Fig. 42, and Supplementary Tables 4 and 5). All-atom MD simulation verified that the grafted multiblock polymers would interact with BSA via hydrophobic interaction and/or hydrogen bonds to some degree (Fig. 4d, e), thereby inducing the burial of BSA surface to different degree (Fig. 4b). In this case, the partial burial of BSA would definitely protect the BSA from thermal degradation, greatly improving the thermostability of BSA bioconjugates. We further performed variable temperature circular dichroism (VT-CD) to investigate the thermal stability of BSA and its bioconjugates (Supplementary Fig. 43). The variable temperature CD spectra ranging from 25  °C to 95  °C were acquired (Supplementary Fig. 43a–g), and the melting temperature (Tm) of BSA and BSA-polymer conjugates was subsequently calculated by analyzing the peak intensity at 209 nm using sigmoid function (Supplementary Fig. 43h). In Supplementary Table 4, all BSA bioconjugates held much higher Tm (>81 oC) than that of BSA itself (70.81 oC), which was consistent with the results in Fig. 3g and Supplementary Table 6, demonstrating that the conjugation of multiblock polymers to BSA greatly improved the thermal stability of the resulting BSA bioconjugates.Structural dynamics of BSA-multiblock polymer conjugatesAll-atom MD simulations was further performed to investigate the structural dynamics of the aforementioned BSA-multiblock polymer conjugates (Fig. 4a). In each model of these six BSA-multiblock polymer conjugates, the grafted multiblock polymer chain maintained an extended conformation using the PACKMOL58 package at the beginning and a 500-ns MD simulation was then conducted based on the initial model for each system (Supplementary Fig. 44). The results showed that the multiblock polymer chain in each system could be quickly contracted and aggregated onto the BSA surface, and ultimately reached equilibrium after around 300 ns (Supplementary Fig. 45a). The extension of the simulation time up to 1 μs for BSA-MMMMMM did not lead to any significant structural rearrangement, suggesting that a 500-ns MD sampling was fairly sufficient (Supplementary Fig. 45b, d). Additionally, the site-selective conjugation of a multiblock polymer chain imposed no profound structural perturbation on BSA (Supplementary Fig. 45c). The last 200-ns MD dataset was adopted for final structural analyses in each system (see Supplementary information).According to the MD results, distinct monomer sequences for these BSA-multiblock polymer conjugates could indeed alter protein/polymer interactions, thereby affecting their binding interfaces. To explore the stability of each BSA-multiblock polymer conjugate, we calculated the buried surface area of BSA upon multipolymer binding and the interaction energies between BSA and multiblock polymer (i.e., electrostatic and van der Waals interactions) by the gmx_MMPBSA59 package. Among the six bioconjugates, BSA-HHHHHH demonstrated the largest buried surface area (~1900 Å2) and the lowest interaction energy (ca. –250 kcal/mol) (Fig. 4b, c), indicating more favorable protein/multiblock polymer contacts compared with other bioconjugates. Further hydrogen-bond analyses verified that BSA-HHHHHH established the most hydrogen-bonds with BSA (Fig. 4d), conferred largely by the hydroxyl group of HEA monomer (H′). Although BSA-MMMMMM and BSA-OOOOOO displayed comparable buried surface area and interaction energies, the latter contained more favorable hydrogen-bond interactions (Fig. 4d) between BSA and hydrophilic multiblock polymer chain. For BSA-MMMMMM, nonpolar contacts were proven to be more critical interactions. Importantly, precise alteration or control over monomer sequence could further finely regulate the protein/polymer interactions. In particular, BSA-MOHMOH and BSA-OMOHHM show more favorable protein/polymer interactions compared with BSA-MMMMMM and BSA-OOOOOO (Fig. 4c). Also, the methyl group of MA monomer (M′) in BSA-MOHMOH exhibited more hydrophobic contacts with BSA surface compared to BSA-MMOOHH and BSA-OMOHHM (Fig. 4e).We further delineated the energy contribution of each monomeric unit along these sequence-controlled multiblock polymer chains, and thereby these six bioconjugates revealed distinct interaction patterns with varied hot-spot binding sites (Fig. 4f). In accordance with the interaction-energy analyses, BSA-HHHHHH exhibited more hot-spot binding sites where a number of hydrogen-bond networks could be formed between BSA and the multiblock polymer via the hydroxyl group in HEA monomer (H′) (Fig. 4f and Supplementary Fig. 46). For BSA-MMMMMM, nonpolar contacts were dominant in protein/polymer interactions, e.g., the MNM61 unit could be inserted into a shallow hydrophobic pocket formed by T507, F508, H509, E564, F567, and A568. For BSA-OOOOOO, the OEG side-chain oxygen atoms in OEGA monomer (O′) could be trapped by positively-charged lysine residue such as BSA-K127 that participated in stabilizing MNO62 and MNO63 (Supplementary Fig. 46). Intriguingly, the above lysine-oxygen interactions were also observed in other BSA-multiblock polymer systems, for example, MNO55 in BSA-MMOOHH, MNO26–27 in BSA-MOHMOH, and MNO10 in BSA-OMOHHM (Supplementary Fig. 46). In addition, diverse hydrogen-bond forms could also be found in BSA-OMOHHM (e.g., via E470, G85, and T83). In addition, the local conformations of active pockets of BSA (i.e., Sudlow sites 1 and 2) were not disturbed (Supplementary Fig. 48a-c), since none of grafted multiblock polymers reached the active pockets of BSA. Furthermore, it was noting that, for the noncovalent BSA/polymer assembly without the covalent linkage, the buried surface area and hydrogen bonds were drastically reduced (Supplementary Fig. 49a–c), indicating that both the covalent linkage and monomer sequence played a key role in the protein/polymer interplay. These results demonstrated, at the atomic level, the specific hot-spot binding sites in both BSA and the multiblock polymers were responsible for regulating the physicochemical properties and biological activities of protein-multiblock polymer conjugates.Sequence-dependent cellular uptakeThe biocompatibility of BSA and BSA-multiblock polymer conjugates was evaluated using MTT assay. In fact, all BSA-multiblock polymer conjugates exhibited much lower cytotoxicity against both L929 and NIH-3T3 cell lines. In Fig. 5b and Supplementary Fig. 50, the cell viability for all BSA bioconjugates almost remained 100% at the concentration of 250 μg/mL or less. Even if the concentration was increased up to 500 μg/mL, the cell viability of BSA bioconjugates was still higher than 90%, indicating the excellent biocompatibility of the resultant BSA-multiblock polymer conjugates. These results demonstrated that the QOILP offered a facile and efficient approach for the fabrication of protein-multiblock polymer conjugates with well-defined structures and sequences, as well as optimal physiochemical and biological properties. The sequence-dependent cellular uptake behaviors (Fig. 5a) of various sequence-controlled BSA-multiblock polymer conjugates with varied sequences in L929 cell lines were investigated by flow cytometry and confocal laser scanning microscopy (CLSM). BSA and BSA-multiblock polymer conjugates were firstly labeled with the NIR dye Cy5.5 enabling real-time monitoring and imaging in vitro, and L929 cells were then incubated with Cy5.5-labeled BSA or its bioconjugates for a range of times up to 4 h. As shown in Fig. 5e, well-distributed fluorescence signal was clearly observed from L929 cells incubated with the BSA-multiblock polymer conjugates at the early stage (0.5 h), whereas negligible fluorescence could be detected from L929 cells incubated with BSA. This result demonstrated that sequence-controlled multiblock polymers greatly facilitated cellular uptake of the resultant bioconjugates by L929 cells. With the extension of incubation time, intracellular fluorescence intensity of L929 cells incubated with BSA or BSA-multiblock polymer conjugates gradually increased, which was confirmed by flow cytometry and CLSM (Fig. 5c and Supplementary Fig. 51), further demonstrating that both BSA and BSA-multiblock polymer conjugates were taken up by cells in a time-dependent manner.Fig. 5: The in vitro cytotoxicity and cellular uptake of BSA-multiblock polymer conjugates by L929 cells.a The illustration of the sequence-dependent cellular-uptake process. b Cytotoxicity assay of BSA and sequence-controlled BSA-multiblock polymer conjugates incubated with L929 cells for 24 h. c, d Cellular uptake of Cy5.5-labeled BSA and Cy5.5-labeled BSA-multiblock polymer conjugates by L929 cells detected by flow cytometry for (c) 30 min and (d) 4 h. Insert: Quantitative fluorescence (FL) intensity comparisons based on the fluorescence-activated cell sorting (FACS). e Confocal images of L929 cells after incubation with Cy5.5-labeled BSA and Cy5.5-labeled BSA-multiblock polymer conjugates for different times (blue: DAPI, red: Cy5.5). Representative images from three independent experiments are shown. The data are shown as mean ± SD (n = 4) for b and the data are shown as mean ± SD (n = 3) for c and d. Figure 5a Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.The effect of monomer sequence of the multiblock polymer on cellular uptake behaviors of BSA-multiblock polymer conjugates was further investigated (Fig. 5c, d and Supplementary Fig. 52). After 0.5 h incubation, the fluorescence intensities of L929 cells incubated with three BSA-multiblock copolymer conjugates (i.e., BSA-MMOOHH, BSA-MOHMOH, and BSA-OMOHHM), were higher than the other three BSA-multiblock homopolymer conjugates (i.e., BSA-MMMMMM, BSA-OOOOOO, and BSA-HHHHHH), in particular, BSA-OMOHHM revealed the fastest cell penetration rate, roughly 2- to 3-fold higher than that of these multiblock homopolymer conjugate systems (Fig. 5c). The variation in cell penetration rate of the bioconjugates might be tentatively attributed to distinct buried surface areas of BSA in each system determined by MD simulations as above (Fig. 4b). After 4 h incubation, L929 cells incubated with BSA-HHHHHH had the strongest fluorescence (Fig. 5c, e), demonstrating highest cell internalization efficiency. It could be attributed to the fact that BSA-HHHHHH showed the maximum hydrogen bonds and the largest buried surface area according to all-atom MD simulations (Fig. 4b, d), which effectively shielded the negative charge of BSA, further facilitating the cellular uptake of the resulting bioconjugate by L929 cells. With alteration in monomer sequence, the fluorescence intensity of BSA-multiblock polymer conjugates increased to varying degrees, disclosing specific sequence-encoded cellular internalization behaviors as depicted in Fig. 5a. Thus, the precision control over the monomer sequence of BSA-multiblock polymer conjugates paved an avenue towards high-efficiency regulation and improvement of the cellular penetration behaviors.Sequence-encoded in vivo biodistribution and pharmacokineticTo further evaluate the effect of monomer sequence on in vivo biodistribution of various sequence-controlled BSA-multiblock polymer conjugates, in vivo near-infrared (NIR) fluorescence imaging was performed in BALB/c mice. After an intravenous injection of Cy5.5-labeled BSA and Cy5.5-labeled BSA-multiblock polymer conjugates, the NIR fluorescence images of BALB/c mice were acquired at predetermined time points (Fig. 6a). At 0.5 h post-injection, the NIR fluorescence was detected throughout the body, and the fluorescence intensity of all BSA-multiblock polymer conjugates in BALB/c mice decayed with time. In vivo biodistribution of BSA and BSA-multiblock polymer conjugates was further investigated by qualitative or quantitative analysis of the fluorescence of excised organs (Fig. 6b, c). The major organs (heart, liver, spleen, lungs, and kidneys) were then harvested after the mice being killed at different injection time intervals. Images of major organs harvested 1 h, 4 h, and 8 h post-injection are depicted in Fig. 6c and Supplementary Fig. 53. The pristine BSA showed much lower mean fluorescence intensity in all organs, suggesting shorter circulatory half-life in vivo ascribed to its biological instability and fast clearance. In contrast, all BSA-multiblock polymer conjugates underwent slower renal clearance and accumulated in liver, kidneys, lung, and spleen. The results indicated that the site-specific conjugation of multiblock polymers to proteins could effectively prolong their circulation time in vivo.Fig. 6: In vivo biodistribution and pharmacokinetic of BSA-multiblock polymer conjugates in BALB/c mice.a In vivo fluorescence images of BALB/c mice at different time intervals after intravenous injection of Cy5.5, Cy5.5-labeled BSA, and Cy5.5-labeled BSA-multiblock polymer conjugates, respectively. b Quantitation analysis of fluorescence intensity in major organs after injection of Cy5.5-labeled BSA and Cy5.5-labeled BSA-multiblock polymer conjugates for 1 h, 4 h, and 8 h. The data are shown as mean ± SD (n = 3). Statistical significance was assessed using a one-way ANOVA with Fisher’s LSD test (*p < 0.05, **p < 0.01, and ***p < 0.001). The exact p values are provided in Source Data file. c Ex vivo images of excised tissues after injection of Cy5.5-labeled BSA and Cy5.5-labeled BSA-multiblock polymer conjugates. d Plasma concentrations of BSA and BSA-multiblock polymer conjugates versus time. The data are shown as mean ± SD (n = 3) for b and d. Source data are provided as a Source Data file.Apparently, these BSA-multiblock polymer conjugates with different monomer sequences displayed distinct levels of enrichment in various organs (Fig. 6c). To accurately visualize the biodistribution of sequence-controlled BSA-multiblock polymer conjugates in various organs of BALB/c mice, we quantified the fluorescence intensity of major organs at different time intervals. In Fig. 6b, the fluorescence intensity declined to varying degrees in various organs as time progressed. In the heart, there was no significant difference in fluorescence intensity among each bioconjugate after 1 h injection, noteworthily, BSA-MMOOHH remained highly enriched over time. Initially, BSA-OOOOOO showed higher enrichment in the liver, while BSA-HHHHHH maintained its enrichment in the liver as time passed. In the spleen, all six BSA-multiblock polymer conjugates showed good enrichment, and all bioconjugates except BSA-MOHMOH experienced attenuation over time. At the early stage of injection, three BSA-multiblock copolymer conjugates had greater fluorescence intensity in the lung than that of BSA-multiblock homopolymer conjugates, but the former decayed more quickly over time than the latter. The three BSA-multiblock copolymer conjugates (i.e., BSA-MMOOHH, BSA-MOHMOH, and BSA-OMOHHM) were less enriched in the kidney than BSA-multiblock homopolymer conjugates, revealing that protein conjugation with sequence-controlled multiblock copolymers could reduce the renal clearance of therapeutic proteins to a great extent. Therefore, it could be concluded that the precision control over monomer sequence could finely regulate protein/polymer interactions in bioconjugates (Fig. 4), further optimizing their biodistribution in vivo.In vivo pharmacokinetics studies of BSA-multiblock polymer conjugates were then conducted by intravenous injection of BSA and BSA-multiblock polymer conjugates in Sprague-Dawley (SD) rats. Figure 6d showed the concentration of BSA and BSA-multiblock polymer conjugates in the plasma over time after injection, and the pharmacokinetic parameters of BSA and its bioconjugates were calculated by fitting the two-compartment model as summarized in Supplementary Table 7. It’s worth noting that all samples were rapidly distributed in SD rat tissues post-injection. Compared to the pristine BSA, the plasma BSA levels of BSA-multiblock polymer conjugates declined slowly over time, as indicated by the longer distribution half-life (t1/2,α). Followed by a rapid distribution phase, BSA and its bioconjugates underwent a slower elimination phase. All BSA-multiblock polymer conjugates had a longer elimination half-life at the beta phase (t1/2,β) than BSA, demonstrating a greatly improved pharmacokinetic profile. Among the BSA-multiblock homopolymer conjugates, BSA-MMMMMM showed a longer terminal half-life (t1/2,β) compared to BSA-OOOOOO and BSA-HHHHHH. In particular, BSA-OMOHHM showed the longest terminal half-life time (t1/2,β) of 18.53 h among all BSA-multiblock polymer conjugates. The difference in pharmacokinetics of BSA-multiblock polymer conjugates might be mainly attributed to their sequence-encoded interaction patterns, morphologies, and bioactivities in vivo.We have illustrated that a facile and robust QOILP synthetic strategy can be successfully applied to the production of sequence-controlled protein-multiblock polymer conjugates with precisely controlled monomer sequence and readily tunable biological functionalities. This quantitative QOILP approach not only simplifies the synthetic technology without isolation of intermediates between each polymerization cycle, but also improves the sequence accuracy, purity, and yield of target protein-multiblock polymer conjugates. All-atom MD simulations were utilized to elucidate the structural dynamics of sequence-controlled protein-multiblock polymer conjugates, further unveiling their sequence-dependent interaction modes and architectures. The precision control over monomer sequence at the molecular level led to a significant enhancement in biostability, esterase-like activity, and biocompatibility of the resultant protein bioconjugates. In vitro and in vivo studies further verified that BSA-multiblock polymer conjugates were endowed with sequence-encoded cellular uptake behaviors, finely regulated in vivo biodistribution, and improved in vivo pharmacokinetics to varying degree. We believe that the QOILP strategy will pave the way to the creation of molecularly-precise, multidimensionally-ordered protein-polymer conjugates with defined monomer sequence, controlled hierarchical structures and tunable functionalities, further enabling the development of next-generation protein therapeutics with maximized therapeutic efficacy as a splendid candidate in disease treatment.
