Synergistic Interplay of Covalent and Non-Covalent Interactions in Reactive Polymer Nanoassembly Facilitates Intracellular Delivery of Antibodies

Abstract

Utility of antibody as a therapeutic is by far limited to extracellular or membrane bound proteins, even though these molecules can be engineered to recognize many “undruggable” therapeutic targets. The primary impediments in developing large antibodies as drugs against intracellular targets involve their low transfection efficiency and suitable reversible encapsulation strategies for intracellular delivery with retention of biological activity. To address this, we outline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies. Through structure-activity studies, we down-select the best performing self-immolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostatics-aided covalent self-assembly approach, we demonstrate efficient encapsulation of medium to large proteins (HRP, 44 kDa and β-gal, 465 kDa) and antibodies (~150 kDa). The designed polymeric nanoassemblies are shown to successfully traffic functional antibodies (anti-NPC and anti-pAkt) to cytosol to elicit their bioactivity towards binding intracellular protein epitopes and inducing apoptosis.

Graphical Abstract

A self-immolative activated carbonate polymer was designed through structure-activity study for efficient protein/antibody encapsulation and redox-sensitive release. Developed polymer-protein/antibody nanoassemblies demonstrated successful intracellular localization of cargoes with retention of cellular activities viz. catalysis or apoptosis.

Introduction

Molecular self-assembly, inspired by recognition processes in nature, has formed the basis for many functional supramolecular architectures.^[1] Although these self-assembled structures are mainly governed by weak non-covalent forces, the co-existence of both covalent and non-covalent interactions is also prevalent in many biological processes. For example, covalent modifications of histones through acetylation and methylation of lysines dictate their electrostatic non-covalent binding interactions with negatively charged DNA in the chromatin structure.^[2] Similarly, in synthetic chemistry the concept of dynamic covalent bonds, coupled with non-covalent templating, has been utilized to create supramolecular structures and to identify ligands for protein targets.^[3] In this article, we report a covalent self-assembly strategy that is templated by non-covalent interactions between the host and the guest molecules to address a key challenge in achieving robust encapsulation of complex and sensitive biomacromolecules.

Intracellular targeting of “undruggable” proteins is a formidable challenge that impacts many diseases with low life expectancy.^[4] Antibodies, long-standing diagnostic candidates in the biologics toolkit, can serve to address this therapeutic challenge as it is now possible to engineer them at large scale for many protein targets.^[5] Unlike small-molecule drugs, antibodies present very high specificity to its target antigens, thus offering therapeutic benefits with minimal side-effects. Binding to a particular epitope via the F_ab region of antibody could turn-off the cellular activity of the protein of interest causing deactivation of relevant biological signaling pathways. In fact, antibody-based therapeutics occupy a large portion of the FDA-approved biologics.^{[5b, 6]} However, this promising class of biologics are so far used for targeting extracellular epitopes and have limited applicability for most intracellular proteins.^{[5d, 7]} This is mainly attributed to the inability of antibodies to penetrate live cell membrane, owing to their large, hydrophilic nature and entrapment in endosomal compartment.^[7–8] Acknowledging the therapeutic need, three key approaches for intracellular delivery of antibody have been taken: (a) physical encapsulation, (b) electrostatic complexation, and (c) covalent conjugation. These strategies have been achieved with peptides, lipids, inorganic or polymer based nanoparticles.^[9] While the carrier mediated strategies, such as liposomal vectors, suffer from low encapsulation efficacy and poor stability, covalent conjugation with polymers often tend to take a toll on the biological activity due to irreversible cargo modifications. Moreover, larger biomacromolecules like antibodies also suffer from poor translocation into the cytosol.^{[5d, 7]}

As a remedy, functional polymers synthesized via controlled polymerization and post-modification techniques have provided many impressive examples for utilization in catalysis, sensing, tissue engineering and controlled drug delivery.^[10] Amongst these, activated ester polymers have gained significant attention for providing enormous flexibility in bioconjugation processes to install a desired functionality, which is otherwise impaired due to the structural instability of sensitive biomolecules under harsh reaction conditions.^{[10a, 11]} Inspired by this, we had designed a self-immolative polymer containing activated carbonate moieties for covalent self-assembly templated by functional proteins through the reactive side chains of polymer.^[12] Lysines, an abundant surface functionality in majority of proteins,^[12–13] had been utilized as conjugation handles for reaction with the activated carbonate moieties to form self-assembled nanostructures. We envisaged the utilization of such reactive covalent self-assembly approach for conjugation of functional antibodies through surface lysines. Due to the presence of reactive side-chain functionalities that are also responsive to redox stimuli, the encapsulated antibodies could be released in a ‘traceless’ manner with retention of its biological activity. However, slow macromolecular reaction kinetics owing to high pK_a of lysine amines,^[14] incomplete reactivity of activated carbonate groups with lysines,^[12] and competitive hydrolytic degradation of polymer are some of the major hurdles for protein conjugation, especially for larger biomacromolecules such as antibodies (~150 kDa).

In this manuscript, we describe the design and synthesis of various activated carbonates containing self-immolative polymers and have studied the kinetics of aminolysis vs. hydrolysis in the context of protein conjugation (Figure 1). A down-selected activated carbonate polymer with higher degree of aminolysis and low hydrolysis is utilized to test protein encapsulation. With the help of an electrostatics-aided covalent capture strategy, we demonstrate efficient encapsulation of large proteins, such as antibodies. Finally, we investigate the cellular delivery of functional antibodies to probe cytosolic localization and evaluated their biological activities in targeting specific intracellular epitopes (Figure 1).

Figure 1.

Schematic representation of an electrostatic-aided covalent self-assembly of polymer network using protein or antibody as the template and its transport into the cytosol in functional form.

Results and Discussion

Synthesis of activated carbonate-containing polymers for protein conjugation.

While the reaction between amines and various activated ester moieties are well-established, including for bioconjugation, such an understanding does not exist for activated carbonates.^{[10a, 11, 15]} Note that utilization of an activated carbonate, instead of the classical activated ester, is critical for reversibility in polymer-protein conjugation through surface lysines. We also envisaged that the resultant carbamate linker would impart hydrolytic stability of the covalent connection, and provide potential biocompatibility due to resemblance to the biologically abundant amide moiety. Stable conjugation with reversible features is critical for a versatile assembly that can translocate the protein across a cellular membrane and release the protein cargo in its native form without any remnants of polymer. To address this, we incorporated a disulfide bond for redox-mediated cleavage at the β-position of the carbonate moiety in the polymer chain. Upon cleavage of the disulfide owing to the presence of higher intracellular glutathione concentration, the self-immolation mechanism will kick-in to release the attached protein tracelessly in its pristine form (Figure 2).^[16] Although we had successfully demonstrated encapsulation of proteins with p-nitrophenyl-carbonate, this functionality fell short of the ability to encapsulate larger proteins, such as antibodies, likely due to low reactivity and competitive hydrolysis issues. We surmised that identifying a reactive functionality that is biased towards aminolysis over hydrolysis would address these challenges.To this end, we synthesized a library of random copolymers containing six potential activated carbonate candidates, viz., nitrophenyl (NPC, previously reported), pentafluorophenyl (PFP), trichlorophenyl (TCL), hexafluoropropanol (CF3), trimethylaminophenyl (NMe3) carbonate moieties (Figure 2a). The polymers were synthesized via RAFT polymerization technique using carbonate methacrylate and PEG methacrylate monomers (Figure 2b–c, see Supporting Information for detailed synthetic procedures). Post-polymerization modification of a PEG-hydroxyethylene disulfide polymer was utilized to synthesize NHS carbonate ester polymer (see Supporting Information).^[12] All monomers and polymers were characterized by NMR (¹H, ¹³C and ¹⁹F NMR (as required)) and gel permeation chromatography. The ratio between the carbonate and PEG groups (~2:8) were evaluated from ¹H NMR (see Supporting Information).

Figure 2.

(a) Schematic of the designed random copolymers for evaluating protein conjugation; (b) General reaction scheme for the synthesis of activated carbonate containing monomers; (c) General reaction scheme for polymerization to achieve random copolymers of PEG and activated carbonate monomers; and (d) Activated carbonate polymer mediated protein conjugation and ‘traceless’ release.

Comparison of aminolysis vs. hydrolysis and efficacy studies for protein conjugation.

To test the designed polymers for conjugation efficacy and degradation kinetics, we first investigated the aminolysis reaction in the presence of a protected small molecule lysine analogue (Figure 3a, S1). Kinetic studies of the desired aminolysis and the competitive hydrolytic degradation were performed by reacting polymers with the lysine analog in phosphate buffer (pH 8.5, previously optimized for protein conjugation).^[12] For TCL-, CF3-, NMe3-, and NPC-polymers, both aminolysis and hydrolysis rates were found to be very slow. Aminolysis rates for PFP- and NHS-polymers were found to be much faster. However, with the NHS-polymer, this increase in rate of aminolysis was also accompanied by a significant increase in the rate of hydrolysis. Considering that the hydrolysis rate in buffer would remain practically unaltered when reacting with macromolecular reactants like proteins compared to small molecule lysine analogue, we hypothesized that the PFP-polymer would perform better for protein conjugation. To investigate the translation of our findings with small molecules on to protein-polymer conjugation, we investigated the reactions of NPC-, PFP-, and NHS-polymers with a model protein, horseradish peroxidase (HRP). Percentage encapsulation of proteins, as evident from the SDS-PAGE analysis, was found to follow the order: PFP (48%) > NHS (18%) > NPC (11%) (Figure 3b). Importantly, to ‘shrink-wrap’ the protein cargo after conjugation, a crosslinking reaction is employed to better protect it from degrading environmental conditions. However, residual activated ester group analyses revealed only ~9% remaining groups for NHS-polymer leaving little room for crosslinking reaction (54% and 90% for PFP and NPC-polymers, Figure 3c). We also found similar bioconjugation efficacy (PFP:54%, NHS: 22% and NPC:18%) for another protein cytochrome C (Figure 3d). These studies further confirm that the PFP-polymer is the appropriate down-selected candidate for protein conjugation.

Figure 3.

(a) Kinetics studies with synthesized activated ester containing polymers to examine extent of aminolysis and hydrolysis reactions; (b) SDS-PAGE analyses for studying HRP encapsulation percentage with the NPC, PFP and NHS-polymers (PFP:48%, NHS:18% and NPC:11%); (c) Measurements of residual activated carbonate moieties left after protein conjugation that can be utilized for crosslinking reaction; (d) SDS-PAGE analyses to show similar encapsulation percentage with Cyt C for the NPC, PFP and NHS-polymers (PFP:54%, NHS: 22% and NPC:18%).

Electrostatics-aided covalent self-assembly strategy for polymer-protein nanoassembly.

In addition to identifying PFP-moiety as the optimal functionality for protein conjugation, we were interested in tuning the structural features further to boost the encapsulation efficacy. Protein surfaces are composed of diverse arrays of amino acids with different surface charges and hydrophobicity. Charged residues on protein surfaces play an important role in dynamic reversible interactions with other biomacromolecules (in protein-protein and protein-antibody complexes).^[17] For most water-soluble proteins, charges are distributed on the surface as a patch with an average size between 1–2 nm.^[18] We hypothesized that introducing a negatively charged group in the activated ester polymer backbone could help recruit protein near the vicinity of polymer via electrostatic interaction with positively charged patches, based on amino acid residues such as lysines, arginines and histidines.^[18–19] We hypothesized that once electrostatically drawn to polymer, the proximity-induced reactivity between lysine functionalities from the protein and the activated carbonate esters on polymer backbone should increase. To test this possibility, we synthesized a random copolymer consisting three different monomeric units, bearing PFP carbonate ester, PEG and 3-sulfopropyl functionalities with a compositional ratio of 2:6:2 (P_Ab, Figure 4a, see Supporting Information). Sulfonate moiety, having pK_a ≈ −0.5,^[20] would remain as negatively charged at the conjugation of pH 8.5, for interactions with positively charged groups on protein surface. To verify the effect of mere electrostatic interactions in protein conjugation, we also synthesized a control polymer consisting PEG and sulfopropyl moieties (SO3-polymer, Figure 4a, also see Supporting Information).

Figure 4.

(a) Design of self-immolative polymer, P_Ab, for protein and antibody conjugation along with control polymers; Polymer-protein nanoassembly characterizations: (b) SDS-PAGE analyses under reducing and non-reducing (with GSH) conditions to show efficient encapsulation and redox-mediated release with the NA-HRP and NA-β-gal nanoassemblies, respectively; (c) Circular dichroism spectra of native HRP, NA-HRP and β-gal, NA-β-gal, respectively; (d) Comparison of protein activity for native, encapsulated and released HRP and β-gal proteins (Encap and Release: NA-Protein nanoassemblies without and with 10 mM GSH treatment, respectively); (e) Intracellular uptake of rhodamine tagged HRP and β-gal delivered with NA-HRP^Rhod and NA-β-gal^Rhod indicating uniform cellular distribution in HeLa cells, respectively; scale bar: 20 μm; and (f) X-gal staining assay showing cytosolic activity of β-gal delivered through NA-β-gal, whereas naked protein sample remained unstained due to cell membrane impermeability.

Before pursuing the antibody conjugation, we tested the encapsulation efficacy and the subsequent stimulus-mediated release of proteins with the designed random copolymer, P_Ab. Two proteins with large molecular weight differences, HRP (~44 kDa) and β-gal (~465 kDa, with four subunits of 116.3 kDa), were chosen to form polymer-protein nanoassembly. While the ζ potential was measured to be similar to the naked protein for NA-HRP (naked HRP: −8 mV; NA-HRP: −9 mV), it was reduced significantly in the case of NA-β-gal (naked β-gal: −25 mV; NA-β-gal: −13 mV) (Figure S2, see Supporting Information). Dynamic light scattering (DLS) measurements showed uniform distribution of nanoassemblies with 119 and 162 nm hydrodynamic diameters for NA-HRP and NA-β-gal, respectively (Figure S2), suggesting efficient shrink-wrapping with crosslinked polymer network. Further analyses with transmission electron microscopy (TEM) revealed discrete nanoassemblies with distribution patterns closely matching with the DLS studies (Figure S3).

To confirm protein encapsulation, we studied gel electrophoresis (SDS-PAGE) under non-reducing conditions. No protein band was observed for nanoassemblies confirming that the proteins were effectively wrapped by the polymer (Figure 4b). Note that the encapsulation efficacies were much higher, ~91% and ~82% for HRP and β-gal respectively, for the P_Ab polymer, compared to the corresponding PFP-polymer without the sulfonate moieties (Figure 3b). Interestingly, control SO3-polymer without the PFP units also showed negligible protein encapsulation (<10%, Figure S4), suggesting that electrostatics alone is not sufficient for efficient encapsulation of proteins either. Thus, it is the combination of electrostatic and covalent self-assembly that offer efficient wrapping of proteins by the polymer. Next, we investigated whether the nanoassembly can release the proteins in a stimuli-responsive manner. The protein bands reappeared in the gel electrophoresis studies, when the nanoassemblies were incubated under reducing conditions (10 mM glutathione, Figure 4b). This concentration corresponds to the typical intracellular GSH concentration of the cytosol.

Retaining the structure and function of the released proteins are critical in developing an effective encapsulation strategy. Towards this goal, we first investigated the secondary structure of the released HRP and β-gal from the nanoassemblies. Circular dichroism spectra of the released proteins showed no apparent changes suggesting conservation of secondary structure of proteins releasing from the nanoassembly upon treatment of glutathione (Figure 4c). Similarly, in vitro activity studies of the released proteins revealed that the proteins activities were greatly silenced (6% and 5% for HRP and β-gal, respectively; Figure 4d, S5–6).^[21] However, upon releasing from the shrink-wrapped state, both proteins regained their enzymatic activities (84% and 87% for HRP and β-gal, respectively). Thus, both structure and functional assays show efficacy of the polymer shell in efficiently wrapping the protein and the recovery of activity of the protein upon encountering a specific environmental stimulus.

Finally, cellular internalization of the protein cargoes was tested for nanoassemblies encapsulated with rhodamine-tagged HRP and β-gal via confocal laser scanning microscopy (CLSM, Figure 4e, S7, see Supporting Information). Uniform red fluorescence in HeLa and MDA-MB-231 cell lines suggested that the nanoassemblies were efficiently internalized by the cells. Furthermore, we were also interested in investigating whether the delivered protein is active, i.e. if the protein was unwrapped to be activated inside the cells. To this end, intracellular activity of delivered β-gal was tested using the x-gal assay. Generation of intense blue color in the cells, compared to the controls, suggests that the protein was not only transported across the cellular membrane to the cytosol, but that it is active inside the cells (Figure 4f and S6).

Extension of the encapsulation strategy to functional antibody.

Inspired by the results with globular proteins, we tested the ability of these polymers to encapsulate and deliver antibodies inside cells. A typical immunoglobulin G (IgG, ~150 kDa) has ~82 surface lysines. Thus, the developed encapsulation strategy with activated carbonate and negatively charged sulfonate moieties could provide chemical and electrostatic handles for boosting the encapsulation of a such large antibody. Indeed, we were able to efficiently form polymer-IgG nanoassemblies (NA-IgG), as evident from the absence of IgG band (at ~150 kDa) for NA-IgG samples in the SDS-PAGE analysis (Figure 5a, S9). TEM and DLS studies revealed monomodal distribution of NA-IgG samples (DLS: 94 nm, TEM: 120 nm), whereas ζ potential was found to be −12 mV (Figure 5b–c, S8 in Supporting Information).

Figure 5.

(a) SDS-PAGE study to show efficient encapsulation of IgG inside the NA-IgG nanoassembly indicated by the absence of IgG band; (b) Transmission electron microscopy images for NA-IgG, scale bar: 500 nm; and (c) Dynamic light scattering measurements for native IgG and NA-IgG nanoassembly; (d) Intracellular uptake of NA-IgGRhod in HeLa cells, scale bar: 20 μm; (e) Mechanism of cellular uptake for NA-IgG^Rhod in presence and absence of endocytic inhibitors; (f) Endosomal colocalization and escape studies after incubation with NA-IgG^Rhod in HeLa cells at 4 and 24 h (green: lysotracker; red: rhodamine B-IgG; blue: nuclear stain, scale bar: 10 μm.

Our ultimate aim is to traffic the antibody for intracellular targeting of specific proteins. Prior to delivering a functional antibody, we were interested in testing the cellular localization of a fluorophore-labelled antibody. A rhodamine labelled IgG was first encapsulated in the polymer nanoassembly (NA-IgG^Rhod) and delivered in HeLa cells. A uniform distribution of red fluorescence, as observed from CLSM studies, confirmed the intracellular access of the delivered IgG (Figure 5d, S10–11, see Supporting Information). In contrast, control polymers (PFP-, SO3- and Gua-polymers) did not show enough cellular red fluorescence, presumably due to low conjugation efficacy of the large cargo (Figure S12). Cellular uptake can be governed by various pathways.^[22] To probe the cellular internalization mechanism for our nanoassemblies, we incubated cells with different endocytosis pathway inhibitors and checked their influence in cellular uptake of nanoparticle via flow cytometry.^[22–23] As evident from Figure 5e and S13, the uptake is governed by the clathrin-mediated endocytosis pathway in HeLa and EMT-6 cells, since hyperosmolar sucrose is the dominant uptake inhibitor. However, all three endocytic pathways (clathrin-, caveolae-mediated and macropinocytosis) were operative in MDA-MB-231 cells. We presume disulfide moieties in the crosslinked polymer backbone could have a major influence in directing the nanoassembly uptake pathway.^[24] Upon endocytosed inside the cells, the next important step for cytosolic access is to escape from the endosome. Time dependent CLSM studies with lysotracker green (an endosome/lysosome marker stain) showed colocalization of red (from rhodamine-labelled IgG) and green (lysotracker) channels after 4 h of incubation with the NA-IgG (Figure 5f, S14). However, separation of red fluorescence clearly indicates that there is a high degree of escape from endosome and cytosolic accumulation of IgG after 24 h. The reasons behind the efficiency of this endosomal escape is not clear to us at this time. As such endosomal escape capabilities are observed with disulfide-based nanoparticles, we provisionally hypothesize that this is related to the literature observations of the interactions between cell surface thiols and disulfide-based macromolecules.^{[24a, 25]} Further, the designed nanoassemblies were also found to be non-toxic even at a high dosage of 2 mg/mL, as revealed from the high cellular viability for NA-IgG samples across three different cell lines (Figure S15).

Intracellular delivery of functional antibodies: anti-pAkt and anti-NPC antibody.

In order to test the potential of polymeric nanoassemblies to deliver antibody inside cells, it is critical to show that the antibody is able to recognize the targeted epitope inside the cells. To this end, we aimed to test the ability of these nanoassemblies in delivering two functional monoclonal antibodies, viz. anti-nuclear pore complex (anti-NPC) and anti-phospho-Akt (Ser473) antibody (Figure 6a). The epitopes for both these antibodies are present in the cytosol and thus analyzing these antibodies would also confirm the endosomal escape that we observed in our fluorescence microscopy studies. When delivered inside cells, anti-NPC antibody bind to the nuclear pore complex located on the nuclear membrane.^{[9b, 9c]} While naked anti-NPC antibody could not penetrate cellular membrane efficiently and failed to locate on the nuclear membrane, NA-anti-NPC could traffic the antibody and successfully highlighted the nuclear pore complex of the cells, evident from the red-colored membrane of the nucleus (stained with a blue dye) due to the binding of the anti-NPC antibody (Figure 6b, S16).

Figure 6.

(a) Schematic of cytosolic delivery for anti-NPC and anti-pAkt antibodies in functional forms through the polymer nanoassembly; (b) Immunostained HeLa cells after NA-anti-NPC delivery showing highlighted nuclear pore complex localized in nuclear membrane, scale: 10 μm; (c) Cellular viability study in MCF-7 cells after delivery of NA-anti-pAkt showing dose dependent decrease in cell survival; (d) Detection of caspase 3/7 in MCF7 cells after NA-anti-pAkt delivery (6 h) using a green fluorescent dye labelled caspase substrate, scale bar: 20 μm; and (e) Western blot analysis showing cleavage of PARP owing to the activation of caspase pathway in MCF-7 cells.

Gratified by this observation, we further tested the intracellular activity with another antibody, anti-pAkt. Protein kinase B, also known as Akt, is an intracellular signal transduction protein responsible for activation of nuclear factor-κB (NF-κB) and several other proteins in the Akt signaling pathway promoting cellular growth.^{[9e, 26]} It inhibits the key apoptotic pathway in many cancer cells and therefore blocking this could result in reinstating the apoptosis mechanism.^[26–27] With this goal in mind, we delivered anti-pAkt antibody with our polymer nanoassembly and were gratified to see that the cellular viability had reduced in a dose-dependent manner (Figure 6c). In addition to utilizing viability as an assay, we were also interested in showing that this is indeed due to the specific inhibition of the Akt pathway. If these results were indeed due to the reactivation of the targeted apoptosis pathway, we should observe the presence of caspase 3/7 enzymes, one of the key controllers for cellular apoptosis pathways. To this end, we examined the presence of caspase 3/7 via immunofluorescence technique. The assay utilizes a caspase substrate attached to a nucleic acid binding dye that only fluoresces upon substrate cleavage by active cellular caspases after binding to nuclear DNA. A clear green fluorescence from the detection assay localized on cellular nucleus confirmed the activation of caspase pathways leading to cellular apoptosis (Figure 6d, S18). To further probe the apoptosis process, we studied the cleavage of poly(ADP-ribose) polymerase (PARP) protein via intracellularly activated caspases through western blot analyses (Figure 6e, S19). The apoptotic activation led to cleavage of PARP (~116 kDa) into 89 and 24 kDa fragments with the NA-anti-pAkt sample, while naked anti-pAkt control did not show any discernible amount of activity. Further, we hypothesize that if the delivered anti-pAkt antibody is efficient in binding the intracellular pAkt protein, its activity will be turned down.^[28] While for untreated cancer cells where the pAkt pathway is upregulated, the pAkt activity should remain high, but the activity should be significantly low for the cells treated with nanoassemblies due to the low availability of unbound active pAkt protein. To test this, we performed an ELISA assay to study pAkt activity in MCF7 cell lysates (Figure S20).^[29] Compared to untreated cells, significant drop in pAkt activities were observed for NA-anti-pAkt (~45%) and positive control (~48%) samples, while native anti-pAkt (~82%) and bare polymer (~86%) were much less efficient. These studies demonstrate the ability of the developed polymeric nanoassembly system in delivering antibody cargoes into the cytosol with the retention of epitope recognition function.

Conclusions

In summary, we demonstrated a versatile strategy for encapsulation of large proteins and antibody therapeutics using an activated carbonate polymer platform. Towards developing a fundamental understanding in the activated carbonate chemistry for protein conjugation, we first developed the structure-reactivity relationship by synthesizing and testing several activated carbonate-bearing polymers for their reactivity in aminolysis vs. hydrolysis. Based on the kinetics studies and preliminary investigation in protein encapsulation experiments, we chose PFP-carbonate as the preferred functionality for further development in encapsulating large proteins, such as antibodies. To further boost the conjugation efficiency, we engineered the polymer structures with electrostatic handles that offered higher degree of encapsulation presumably through proximity-induced reactivity enhancement. We show here that the electrostatics-aided covalent encapsulation strategy provided a robust platform to (i) capture larger antibody molecules with high fidelity that are found difficult to encapsulate otherwise; (ii) protect the structure and silence the cargo activity while in encapsulated state; (iii) enable regaining the functional activity of the payload upon release by the influence of an intracellular stimulus; (iv) efficiently deliver the encapsulated cargo into the cytosol; and (v) ensure that the desired biological functions of the cargo are retained upon intracellular delivery and cytosolic release. The developed polymer nanoassembly system could serve as a promising protein delivery platform, specifically for antibody-based intracellular drug targets, which is so far considered as one of the most challenging goals for the development of antibody therapeutics.