Peptide-polymer Conjugates Research Articles

Precise Control of Self‐assembly in vivo Based on Polymer‐Peptide Conjugates†

Comprehensive SummaryThe self‐assembled nanomaterials based on peptides, which have not only good biocompatibility and low toxicity but also high stability and performance, are increasingly becoming an important way for cancer treatment. In this review, we highlight the progress of in vivo self‐assembled polymer‐peptide conjugates (PPCs) and their application, showing the recent advances in the development of “in vivo self‐assembly” strategy for cancer treatment. Given the diverse microenvironments of tumor cells, different responsive assembly strategies (enzyme, pH, redox, temperature, etc.) have been developed to precisely control the assembly at different biological levels, realizing enhanced drug delivery and improved anticancer efficacy.

Organic Mixed Ion-Electron Conductivity in Polymer Hybrid Systems.

Recently, organic materials with mixed ion/electron conductivity (OMIEC) have gained significant interest among research communities all over the world. The unique ability to conduct ions and electrons in the same organic material adds to their use in next generation electrochemical, biotechnological, energy generation, energy storage, electrochromic, and sensor devices. Semiconducting conjugated polymers are well-known OMIECs due to their feasibility for both ion and electron transport in the bulk region. In this mini-review, we have shed light on conjugated polymers with ionic pendent groups, block copolymers of electronically and ionic conducting polymers, polymer electrolytes, blends of conjugated polymers with polyelectrolyte/polymer electrolytes; blends of conducting polymer with small organic molecules including conducting polymer–peptide conjugates; and blends of nonconjugated polymers as mixed conducting systems. These systems not only include the well-studied OMEIC systems, but also include some new systems where the OMEIC property has been predicted from the typical current–voltage (I–V) plots. The conduction mechanism of ions and electrons, ion-electron coupling, directionality, and dimensionality of these OMEIC materials are discussed in brief. The different properties of OMEIC materials and their applications in diverse fields like energy, electrochromic, biotechnology, sensing, and so forth are enlightened together with the perspective for future improvement of OMEIC materials.

Unraveling On-demand Strain-Stiffening in Nanofibrous Peptide–Polymer Conjugates to Mimic Contractility in Actinomyosin Networks

Reinforcement owing to inner stress formation is of supreme importance in life and is the basis for biomechanical pathways, especially in the contractile materials resembling muscles. Cells strengthen their contiguous matrix by pulling thin actin filaments associated with bundles of myosin with molecular motors promoting rapid fluidization and dynamic stiffening of the cytoskeleton. Herein, we demonstrate an elegant synthetic design of a peptide–polymer conjugate network that exhibits multiple hierarchical control over its stiffening. Dynamic Schiff base crosslinking of semi-flexible peptide nanofibers with the thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) copolymer endows a covalent network. Further, the photo-dimerizable 4-methylcoumarin moieties at the core of peptide nanofibers can also be reversibly photo-fixated with the choice of light. The conjugates exhibit macroscopic heat-stiffening response by generating inner stress through a coil-to-globule transition owing to the lower critical solution temperature of PNIPAM. Moreover, the covalently crosslinked network noticeably stiffens in response to applied shear stress that can be further ramped up in the photo-fixated peptide nanofibers. Finally, an excellent biocompatibility toward U2OS cell lines validates these as ideal biomimetic and adaptive materials. Overall, we report for the first time a synthetic strain-stiffening network based on a non-equilibrium self-assembled peptide fiber toward non-linear biomechanics analogous to the actinomyosin network ubiquitous in cells and tissues.

Rational Design of Hydrogel Networks with Dynamic Mechanical Properties to Mimic Matrix Remodeling.

Engineered hydrogels are increasingly used as extracellular matrix (ECM) surrogates for probing cell function in response to ECM remodeling events related to injury or disease (e.g., degradation followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo-reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically triggered crosslink degradation, and light-triggered photopolymerization stiffening. Hydrogels are formed by a photo-initiated thiol-ene reaction between multiarm polyethylene glycol and a dually enzymatically degradable peptide linker, which incorporates a thrombin-degradable sequence for triggered softening and a matrix metalloproteinase (MMP)-degradable sequence for cell-driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP-degradable polymer-peptide conjugate and multiarm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E) ≈5 to 1.5 to 6kPa with >3x ΔE).The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation shows a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible.

Polymer chimera of stapled oncolytic peptide coupled with anti-PD-L1 peptide boosts immunotherapy of colorectal cancer

Rationale: Scarce tumor mutation burden and neoantigens create tremendous obstacles for an effective immunotherapy of colorectal cancer (CRC). Oncolytic peptides rise as a promising therapeutic approach that boosts tumor-specific immune responses by inducing antigenic substances. However, the clinical application of oncolytic peptides has been hindered because of structural instability, proteolytic degradation, and undesired toxicity when administered systemically.Methods: Based on wasp venom peptide, an optimized stapled oncolytic peptide MP9 was developed with rigid α-helix, protease-resistance, and CRC cell cytotoxicity. By incorporating four functional motifs that include D-peptidomimetic inhibitor of PD-L1, matrix metalloproteinase-2 (MMP-2) cleavable spacer, and MP9 with 4-arm PEG, a novel peptide-polymer conjugate (PEG-MP9-aPDL1) was obtained and identified as the most promising systemic delivery vehicle with PD-L1 targeting specificity and favorable pharmacokinetic properties.Results: We demonstrated that PEG-MP9-aPDL1-driven oncolysis induces a panel of immunogenic cell death (ICD)-relevant damage-associated molecular patterns (DAMPs) both in vitro and in vivo, which are key elements for immunotherapy with PD-L1 inhibitor. Further, PEG-MP9-aPDL1 exhibited prominent immunotherapeutic efficacy in a CRC mouse model characterized by tumor infiltration of CD8+ T cells and induction of cytotoxic lymphocytes (CTLs) in the spleens.Conclusion: Our findings suggest that PEG-MP9-aPDL1 is an all-in-one platform for oncolytic immunotherapy and immune checkpoint blockade (ICB).

Tailoring the luminescence of FRET systems built using supramolecular polymeric nanotubes

Supramolecular polymeric nanotubes self-assembled from cyclic peptide–polymer conjugates are employed as general scaffolds to fabricate supramolecular FRET systems with tailorable and responsive luminescence.

Well-Defined Mannosylated Polymer for Peptide Vaccine Delivery with Enhanced Antitumor Immunity.

Peptide-based cancer vaccines offer production and safety advantages but have had limited clinical success due to their intrinsic instability, rapid clearance, and low cellular uptake. Nanoparticle-based delivery vehicles can improve the in vivo stability and cellular uptake of peptide antigens. Here, a well-defined, self-assembling mannosylated polymer is developed for anticancer peptide antigen delivery. The amphiphilic polymer is prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the peptide antigens are conjugated to the pH-sensitive hydrophobic block through the reversible disulfide linkage for selective release after cell entry. The polymer-peptide conjugates self-assemble into sub-100nm micelles at physiological pH and dissociate at endosomal pH. The mannosylated micellar corona increases the accumulation of vaccine cargoes in the draining inguinal lymph nodes and facilitates nanoparticle uptake by professional antigen presenting cells. In vivo studies demonstrate that the mannosylated micelle formulation improves dendritic cell activation and enhances antigen-specific T cell responses, resulting in higher antitumor immunity in tumor-bearing mice compared to free peptide antigen. The mannosylated polymer is therefore a simple and promising platform for the delivery of peptide cancer vaccines.

Tumor Marker B7-H6 Bound to the Coiled Coil Peptide-Polymer Conjugate Enables Targeted Therapy by Activating Human Natural Killer Cells

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

Development of Polythiophene–Tripeptide Covalent Conjugates Showing Excellent Structure-Dependent Photophysical and Photocurrent Properties

Presently, conducting polymer–peptide conjugates have drawn great interest for their interesting optoelectronic and photophysical properties. To understand the structural dependence of the conjugates on their properties, we have covalently coupled poly(3-thiophene acetic acid) (P3TAA) with a C-protected tripeptide, H2N-F-F-V-OMe (TPEP), to produce their conjugate PTCP-I. A carboxylic acid-deprotected tripeptide polythiophene conjugate (PTCP-II) is also produced by alkaline hydrolysis of PTCP-I to delineate any difference in properties due to the change in molecular structure. The stretching frequencies for −NH– and >C═O groups of PTCP-II are 11 and 26 cm–1 lower than those of PTCP-I, indicating better intermolecular hydrogen bonding interactions in the former. This alters the seaweed morphology of PTCP-I to a sheet morphology for PTCP-II. The blue shift of the π–π* absorption band of polythiophene with gradually increasing the PTCP-I concentration is attributed to the H-type aggregation in DMF solution, but in the solid state, this peak shows a 17 nm red shift from its solution state for better electron delocalization for closer proximity of molecules. The UV–Vis peak of PTCP-II shows a lower blue shift than that of PTCP-I in DMF, attributed to the uncoiled polythiophene chains of the former. In DMF solution, interestingly, both PTCP-I and PTCP-II conjugates exhibit aggregation-induced emission (AIE), showing maxima at 0.08 mg/mL and then aggregation-induced quenching (AIQ) with further increasing its concentration. PTCP-I and PTCP-II exhibit dc conductivities of 0.04 and 0.55 μS cm–1, respectively, at 30 °C, and the higher value for PTCP-II is due to its lower band gap (1.91 eV) compared to that of PTCP-I (2.14 eV). The current (I)–voltage (V) plot of both conjugates exhibits semiconducting properties, which are higher for PTCP-II for its lower band gap. The photocurrent behavior of the conjugates indicates that the on–off response in the PTCP-II system is significantly higher than that in PTCP-I due to the unfolded polymer chain of the former, as the photogenerated electrons and holes can move more freely there. Impedance spectra of both the conjugates show single semicircles for the Nyquist plots, and the charge transfer resistance of PTCP-II is 4.4 times lower than that of PTCP-I but the capacitance value is 27 times higher, arising from the more extended chain configuration of the former, signifying structural importance on the optoelectronic and photocurrent properties of the polymer–peptide conjugates.

Novel Peptide–Polymer Conjugate with pH-Responsive Targeting/Disrupting Effects on Biomembranes

Conjugating polymers to peptides has become a new strategy of designing functional antitumor agents for their improved stability and enhanced activity. In this paper, a novel peptide-polymer conjugate PEPc-PMAA with pH responsiveness was designed and synthesized. The isoelectric point of PEPc was studied by dynamic light scattering for the targeting effect. Also, the transmittances of PMAA at different pHs were measured using an ultraviolet-visible spectrophotometer for determining the triggering pH of the disrupting effect. The results showed that PEPc-PMAA was hydrophilic under neutral conditions and changed to be amphiphilic composed of positively charged PEPc and hydrophobic PMAA under acidic conditions. The interactions between PEPc-PMAA and mimic cells were investigated by the measurements of membrane fluidity and cargo leakage from 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and 1,2-dipalmitoyl-sn-glycerol-3-phospho-(1-rac-glycerol) (DPPG) liposomes. It proved that PEPc-PMAA caused a distinct membrane disturbance of the DPPG liposome at pH 5.5, resulting in more serious cargo leakage. Because of its targeting and disrupting effects on negatively charged biomembranes under acidic conditions, PEPc-PMAA showed its good potential as an antitumor agent.

Polyacrylate-GnRH Peptide Conjugate as an Oral Contraceptive Vaccine Candidate.

Contraceptive vaccines are designed to elicit immune responses against major components of animal reproductive systems. These vaccines, which are most commonly administered via injection, typically target gonadotropin-releasing hormone (GnRH). However, the need to restrain animals for treatment limits the field applications of injectable vaccines. Oral administration would broaden vaccine applicability. We explored contraceptive vaccine candidates composed of GnRH peptide hormone, universal T helper PADRE (P), and a poly(methylacrylate) (PMA)-based delivery system. When self-assembled into nanoparticles, PMA-P-GnRH induced the production of high IgG titers after subcutaneous and oral administration in mice. PADRE was then replaced with pig T helper derived from the swine flu virus, and the vaccine was tested in pigs. High levels of systemic antibodies were produced in pigs after both injection and oral administration of the vaccine. In conclusion, we developed a simple peptide–polymer conjugate that shows promise as an effective, adjuvant-free, oral GnRH-based contraceptive vaccine.

Computational design of a minimal “protein-like” conjugate for potent membrane poration

Biomimetic membrane nanopores have great potentials for bio- and nanotechnology applications. Here, a design strategy of an artificial protein by reasonably conjugating peptides and polymers for membrane poration was developed based on the α-helical bundle framework. A serial of peptide-polymer conjugates, composed of the α-helical peptide melittin (Mel) and clinically available polymer polyethylene glycol, were fabricated. Within them, the S-Mel, consisting of four helices arranged in a star-like bundle structure, demonstrated the most improved performance. Specifically, one single S-Mel molecule can build a stable membrane pore. Moreover, the obtained pores display stimuli-responsive properties in both size (including water flux) and structure, which is ascribed to the molecular architecture-regulated helical packing changes of S-Mel during membrane actions. These results promise S-Mel the ability to achieve ONOFF switching and gating effects for selective mass translocation across a cell membrane and help understand the structure-function relationship of proteins.

Molecular Self-Assembly and Supramolecular Chemistry of Cyclic Peptides.

This Review focuses on the establishment and development of self-assemblies governed by the supramolecular interactions between cyclic peptides. The Review first describes the type of cyclic peptides able to assemble into tubular structures to form supramolecular cyclic peptide nanotubes. A range of cyclic peptides have been identified to have such properties, including α-peptides, β-peptides, α,γ-peptides, and peptides based on δ- and ε-amino acids. The Review covers the design and functionalization of these cyclic peptides and expands to a recent advance in the design and application of these materials through their conjugation to polymer chains to generate cyclic peptide–polymer conjugates nanostructures. The Review, then, concentrates on the challenges in characterizing these systems and presents an overview of the various analytical and characterization techniques used to date. This overview concludes with a critical survey of the various applications of the nanomaterials obtained from supramolecular cyclic peptide nanotubes, with a focus on biological and medical applications, ranging from ion channels and membrane insertion to antibacterial materials, anticancer drug delivery, gene delivery, and antiviral applications.

Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering.

Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization.

Replacement of L-amino acid peptides with D-amino acid peptides mitigates anti-PEG antibody generation against polymer-peptide conjugates in mice

The generation of anti-PEG antibodies in response to PEGylated proteins, peptides, and carriers significantly limits their clinical applicability. IgM antibodies mediate the clearance of these therapeutics upon repeat injection, resulting in toxicity and hindered therapeutic efficacy. We observed this phenomenon in our polymer platform, virus-inspired polymer for endosomal release (VIPER), which employs pH-sensitive triggered display of a lytic peptide, melittin, to facilitate endosomal escape. While the polymer-peptide conjugate was well tolerated after a single injection, we observed unexpected mortality upon repeat injection. Thus, the goal of this work was to enhance the safety and tolerability of VIPER for frequent dosing. Based on previous reports on anti-PEG antibodies and the adjuvant activity of melittin, we characterized the antibody response to polymer, peptide, and polymer-peptide conjugates after repeat-dosing and measured high IgM titers that bound PEG. By substituting the L-amino acid peptide for its D-amino acid enantiomer, we significantly attenuated the anti-PEG antibody generation and toxicity, permitting repeat-injections. We attempted to rescue mice from L-melittin induced toxicity by prophylactic injection of platelet activating factor (PAF) antagonist CV-6209, but observed minimal effect, suggesting that PAF is not the primary mediator of the observed hypersensitivity response. Overall, we demonstrated that the D-amino acid polymer-peptide conjugates, unlike L-amino acid polymer-peptide conjugates, exhibit good tolerability in vivo, even upon repeat administration, and do not elicit the generation of anti-PEG antibodies.

Tubular supramolecular alternating copolymers fabricated by cyclic peptide-polymer conjugates.

Supramolecular copolymers are an emerging class of materials, which bring together different properties and functionalities of multiple components via noncovalent interactions. While it is widely acknowledged that the repeating unit sequence plays an essential role on the performance of these materials, mastering and tuning the supramolecular copolymer sequence is still an open challenge. To date, only statistical supramolecular copolymers have been reported using cyclic peptide–polymer conjugates as building blocks. To enrich the diversity of tubular supramolecular copolymers, we report here a strategy of controlling their sequences by introducing an extra complementary noncovalent interaction. Hence, two conjugates bearing one electron donor and one electron acceptor, respectively, are designed. The two conjugates can individually assemble into tubular supramolecular homopolymers driven by the multiple hydrogen bonding interactions between cyclic peptides. However, the complementary charge transfer interaction between the electron donor and acceptor makes each conjugate more favorable for complexing with its counterpart, resulting in an alternating sequence of the supramolecular copolymer. Following the same principle, more functional supramolecular alternating copolymers are expected to be designed and constructed via other complementary noncovalent interactions (electrostatic interactions, metal coordination interactions, and host–guest interactions, etc.).

Molecular engineering of antimicrobial peptide (AMP)-polymer conjugates.

As antimicrobial resistance becomes an increasing threat, bringing significant economic and health burdens, innovative antimicrobial treatments are urgently needed. While antimicrobial peptides (AMPs) are promising therapeutics, exhibiting high activity against resistant bacterial strains, limited stability and toxicity to mammalian cells has hindered clinical development. Attaching AMPs to polymers provides opportunities to present AMPs in a way that maximizes bacterial killing while enhancing compatibility with mammalian cells, stability, and solubility. Conjugation of an AMP to a linear hydrophilic polymer yields the desired improvements in stability, mammalian cell compatibility, and solubility, yet often markedly reduces bactericidal effects. Non-linear polymer architectures and supramolecular assemblies that accommodate multiple AMPs per polymer chain afford AMP-polymer conjugates that strike a superior balance of antimicrobial activity, mammalian cell compatibility, stability, and solubility. Therefore, we review the design criteria, building blocks, and synthetic strategies for engineering AMP-polymer conjugates, emphasizing the connection between molecular architecture and antimicrobial performance to inspire and enable further innovation to advance this emerging class of biomaterials.

Comparative Study of the Cellular Uptake and Intracellular Behavior of a Library of Cyclic Peptide-Polymer Nanotubes with Different Self-Assembling Properties.

Particle shape has been described as a key factor in improving cell internalization and biodistribution among the different properties investigated for drug-delivery systems. In particular, tubular structures have been identified as promising candidates for improving drug delivery. Here, we investigate the influence of different design elements of cyclic peptide–polymer nanotubes (CPNTs) on cellular uptake including the nature and length of the polymer and the cyclic peptide building block. By varying the composition of these cyclic peptide–polymer conjugates, a library of CPNTs of lengths varying from a few to over a 150 nm were synthesized and characterized using scattering techniques (small-angle neutron scattering and static light scattering). In vitro studies with fluorescently labeled CPNTs have shown that nanotubes comprised of a single polymer arm with a size between 8 and 16 nm were the most efficiently taken up by three different mammalian cell lines. A mechanistic study on multicellular tumor spheroids has confirmed the ability of these compounds to penetrate to their core. Variations in the proportion of paracellular and transcellular uptake with the self-assembling potential of the CPNT were also observed, giving key insights about the behavior of CPNTs in cellular systems.

Characterizing aggregate growth and morphology of alanine-rich polypeptides as a function of sequence chemistry and solution temperature from scattering, spectroscopy, and microscopy

The aggregation behavior and stability of a series of alanine-rich peptides, which are included as components of peptide-polymer conjugates, were characterized using a combination of biophysical techniques. Light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. The results show large particles immediately upon dissolution in buffer. At room temperature, these particles relaxed to reach a mostly monomeric peptide state, while at higher temperatures, they grew to form aggregates. Circular dichroism spectroscopy (CD) was used to monitor temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all of the sequences are helical at low temperatures with transitions to non-helical conformation with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, the shorter and longer peptide sequences showed notable changes in conformation, and were not able to recover their original helicity after 72 h. After incubation for up to one week, β-sheet conformations were observed in these two cases, while only α-helical conformation loss was observed for the peptide of intermediate molecular weight. Transmission electron microscopy measurements reveal the formation of fibrils after 72 h of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation can be stalled when solutions are cooled to room temperature.

Antimicrobial Polymer-Peptide Conjugates Based on Maximin H5 and PEG to Prevent Biofouling of E. coli and P. aeruginosa.

Many pathogens, such as Pseudomonas aeruginosa and Escherichia coli bacteria can easily attach to surfaces and form stable biofilms. The formation of such biofilms in surfaces presents a problem in environmental, biomedical, and industrial processes, among many others. Aiming to provide a plausible solution to this issue, the anionic and hydrophobic peptide Maximin H5 C-terminally deaminated isoform (MH5C) has been modified with a cysteine in the C-terminal (MH5C-Cys) and coupled to polyethylene glycol (PEG) polymers of varying sizes (i.e., 2 kDa and 5 kDa) to serve as a surface protective coating. Briefly, the MH5C-Cys was bioconjugated to PEG and purified by size exclusion chromatography while the reaction was confirmed via SDS-PAGE and MALDI ToF. Moreover, the preventive antimicrobial activity of the MH5C-Cys-PEG conjugates was performed via the growth curves method, showing inhibition of bacterial growth after 24 h. The efficacy of these peptide-polymer conjugates was extensively characterized via scanning electron microscopy (SEM), minimum inhibition concentration (MIC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC) assays to evaluate their ability to eradicate and prevent the biofilms. Interestingly, this work demonstrated a critical PEG polymer weight of 5 kDa as ideal when coupled to the peptide to achieve inhibition and eradication of the biofilm formation in both bacteria strains. According to the MICs (40 μM) and MBICs (300 μM), we can conclude that this conjugate (MH5C-Cys-5 kDa) has an action that prevents/inhibits the formation of biofilms and the eradication of biofilms (MBEC 500 μM). In contrast, the MH5C-Cys peptide with PEG polymer of 2 kDa did not show inhibition or eradication of the biofilms.