Non-Genetic Cell Surface Engineering

• Non-Genetic Cell Surface Engineering is a set of methods that functionalizes cell surfaces using synthetic molecules, nanostructures, or modified substrates, while preserving the genomic integrity of cells.
• Techniques such as substrate lithography, PBS and plasma treatments, and direct membrane conjugation enable precise modulation of cell adhesion, signaling, and mechanical properties.
• Applications span tissue engineering, biosensing, and IoBNT, providing reversible, programmable, and eco-friendly alternatives to traditional genetic modifications.

Non-Genetic Cell Surface Engineering (NG-CSE) refers to a set of methodologies for functionalizing the surfaces of living cells by attaching synthetic molecules, nanostructures, or modifying substrates, all without altering cellular genomic DNA. NG-CSE confers programmable, reversible, and spatially precise control over cell behavior, adhesion, signaling, and extrinsic functionalities. Techniques span from substrate nanofabrication and physicochemical treatments to direct conjugation of synthetic molecular devices, enabling a vast landscape of applications in tissue engineering, biosensing, molecular communications, and the Internet of Bio-Nano Things (IoBNT).

1. Physical and Chemical Principles Underlying NG-CSE

NG-CSE exploits the cell membrane's role as the primary interface between cells and their external milieu. Changes in surface stiffness, energy, chemical functionality, and nano-topography critically influence cell adhesion, migration, and cytoskeletal organization (Martini et al., 2013). Substrate modification is a foundational approach: surface energy is tailored via chemical end-groups (hydrophilic vs. hydrophobic), while nano-patterns and topographical features—on scales comparable to focal adhesion complexes (90–500 nm)—precisely dictate cellular responses.

Mechanical signaling through substrate stiffness is quantitatively accessible using AFM-based Hertzian contact mechanics:

$F = \frac{4}{3} E \sqrt{R}\, \delta^{3/2}$

where $F$ is the applied force, $E$ the cell’s Young modulus, $R$ the probe radius, and $\delta$ the indentation depth.

Chemical functionalities, e.g., –CF₃ (FDTS) or –NH₂ (APTES), control hydrophilicity and protein adsorption, which subsequently modulate receptor binding and downstream cell behavior. This enables the engineering of surfaces that selectively promote or inhibit cell contact in patterned regions.

2. Methodologies for NG-CSE

Surface Patterning and Lithographic Techniques

Polymer Blend Lithography (PBL) (Martini et al., 2013) is an advanced method for producing nanopatterned surfaces with high chemical and biofunctional contrast. Major steps include:

• Spin-coating immiscible polymer blends under controlled humidity
• Selective dissolution (e.g., acetic acid) removes one phase, leaving a mask
• Vapor-phase deposition of self-assembled monolayers (SAMs) (FDTS or APTES) functionalizes exposed regions
• Mask removal yields a surface with submicron lateral chemical domains

This bottom-up approach, driven by phase separation and self-organization, enables fine-tuning of topography and chemical cues on length scales matching cell adhesion complexes.

Chemical and Physical Treatments

Phosphate buffered saline (PBS) immersion modifies acrylate-based polymeric scaffolds for enhanced epithelial cell adhesion (Santos et al., 27 May 2024). PBS treatment induces:

• Increased hydrophilicity (contact angle shift from 64.93° to 41.48°, S59/PBS)
• Introduction of hydroxyl and carboxyl functional groups (verified by FTIR: peaks at 1370 cm⁻¹, 1760 cm⁻¹, and 1714 cm⁻¹)
• Modulation of surface roughness, with the ratio $r \approx 1.0052$ correlating with higher adhesion

These modifications promote protein adsorption and cell spreading without altering cell machinery.

Low-temperature plasma treatment (Rondøn et al., 4 Apr 2025) similarly introduces –OH, –COOH, and –C=O groups, increases surface energy (optimal contact angle ∼70° via Young’s equation), and enhances nano-/micro-scale roughness, collectively improving cell proliferation and anchorage. Plasma-induced radicals also permit covalent immobilization of bioactive molecules.

Direct Cell Membrane Engineering

Conjugation of nanostructures (e.g., DNA nanotubes) to cell surface receptors employs antibody-mediated DNA-directed attachment (AMDA) (Jia et al., 2021):

• Cell-surface receptors are labeled with antibody-biotin complexes
• DNA tags (BDC) are anchored via streptavidin bridges
• Seed nanostructures with complementary DNA (BDC′) rapidly hybridize at the cell surface (rate constants $10^5$–$10^6$ M⁻¹s⁻¹)
• The technique achieves spatial and kinetic control, with the nanodevice anchored precisely at selected membrane loci

Further, synthetic components may be attached via covalent (NHS ester reactions) or non-covalent (hydrophobic insertion of cholesterol/lipid-conjugated molecules) chemistries (Ince et al., 21 Sep 2025).

3. Functional Outcomes and Device Integration

NG-CSE enables precise modulation of cellular functions and behavior:

• Patterned substrates guide cell adhesion, migration velocities, and cytoskeletal restructuring (Martini et al., 2013)
• PBS and plasma treatments result in 3x increases in adhesion on optimized surfaces, with cell morphology (shape factor ∼0.56, aspect ratio ∼0.34) closely mirroring physiological states (Santos et al., 27 May 2024)
• Anchored DNA nanotubes function as shear stress meters, displaying quantitative bending responses (torque $M = -½ a μ U L^2 \sin(θ)$) to applied fluid flows in the physiological regime (0–2 dyn/cm²) (Jia et al., 2021)
• Dynamic growth and end-to-end joining of DNA nanotubes allow for real-time reconfigurable device architectures on the cell membrane

Surface engineering directly impacts the design of tissue engineering scaffolds, biosensors, and micro-physiological systems by creating controlled environments for cell assembly and interface.

4. NG-CSE in the Internet of Bio-Nano Things (IoBNT)

NG-CSE uniquely positions living cells as programmable, functional nodes ("Bio-Nano Things" or BNTs) in IoBNT architectures (Ince et al., 21 Sep 2025). Strategies for cell "hijacking" include:

• Covalent grafting of synthetic modules to membrane amines via NHS-ester chemistry
• Non-covalent insertion of lipid-anchored functional groups
• Installation of programmable DNA nanodevices, logic gates, and sensors via surface-tethered aptamer circuits, described by strand displacement kinetics:

$$\frac{d[\text{Complex}]}{dt} = k_{\text{on}} [\text{Aptamer}] [\text{Trigger}] - k_{\text{off}} [\text{Complex}]$$

• Modular surface functionalization with quantum dots, Fe₃O₄ nanoparticles, PEG, and targeted peptides

Advantages over genetic modification include transientness, reversibility, and preservation of intrinsic cellular functions, minimizing safety concerns and metabolic burdens.

Emerging IoBNT architectures enabled by NG-CSE include:

Architecture	Principle	Application
Circulating Sentinel Networks	In-situ RBC hijacking	Liquid biopsy, surveillance
In Vitro Biocomputers	DNA zip-code assembly	Cellular computation

Cells are exploited both as engineered devices and autonomous agents, supporting programmable communication, actuation, and network topology reconfiguration.

5. Challenges and Future Directions

Dynamic turnover of cell-surface modifications (membrane recycling, lateral diffusion, endocytosis) sources time dependence in molecular communication channels. This necessitates multi-scale modeling:

• Nanoscale: synthesis, binding, and decay kinetics
• Cellular scale: membrane dynamics, stochastic turnover
• Populational scale: agent-based models to capture emergent network behavior (Ince et al., 21 Sep 2025)

Research focuses on developing reversible, stimuli-responsive linkers (dynamic covalent chemistry), integrating multi-scale data into predictive computational models, and designing agency-aware network protocols that exploit cellular richness instead of treating cells as passive substrates.

A plausible implication is that the evolution of NG-CSE will rely on precision surface engineering, integration of smart functional materials, and advances in computational frameworks to fully exploit cellular agency and programmability in living bio-cyber systems.

6. Implications for Biomedical Engineering and Regenerative Medicine

NG-CSE advances the construction of next-generation tissue engineering scaffolds, implantable biomedical devices, and biosensors by providing robust, reproducible, and scalable means of modulating cell adhesion, spreading, and network formation (Santos et al., 27 May 2024, Rondøn et al., 4 Apr 2025). The eco-friendly and low-cost nature of PBS and plasma treatments further enables sustainable manufacturing.

In regenerative medicine, fine-tuned scaffold properties offer customizable environments for cell differentiation and tissue formation, bypassing complications of genetic modification. The toolbox of NG-CSE can be extended with bioactive molecule immobilization for dynamic and responsive cell–biomaterial interactions.

This suggests that continued refinement of non-genetic cell surface engineering methodologies will underpin future progress in biofunctional devices, advanced tissue models, and integrated bio-nano networks.