Biofilm-associated infections pose a significant challenge in clinical settings due to their inherent resistance to conventional antimicrobial therapies. The extracellular polymeric substance (EPS) matrix, a key component of biofilms, acts as a physical and chemical barrier that limits drug penetration and shields embedded microbial communities. Enzymatic disruption of the EPS, combined with antimicrobial agents, has emerged as a promising strategy to enhance biofilm eradication. Nanoparticle systems co-loaded with enzymes such as DNase and proteinase K, alongside antimicrobials, offer a robust solution by protecting enzymatic activity and improving delivery to the biofilm core.

The EPS is composed of polysaccharides, proteins, extracellular DNA (eDNA), and lipids, which contribute to biofilm structural integrity and resistance. DNase degrades eDNA, a critical component for biofilm stability, while proteinase K hydrolyzes proteins within the EPS. However, free enzymes face rapid degradation in physiological environments and poor penetration into dense biofilm matrices. Nanocarriers address these limitations by encapsulating enzymes, shielding them from proteolytic degradation, and facilitating their transport through the EPS. Polymeric nanoparticles, liposomes, and inorganic nanocarriers have been explored for this purpose, each offering distinct advantages in stability, loading capacity, and release kinetics.

Polymeric nanoparticles, such as those made from poly(lactic-co-glycolic acid) (PLGA), are widely used due to their biocompatibility and controlled release properties. For instance, PLGA nanoparticles co-loaded with DNase and the antibiotic vancomycin demonstrated enhanced penetration into Staphylococcus aureus biofilms. The nanoparticles protected DNase from serum proteases, maintaining over 80% enzymatic activity after 24 hours in serum-containing media. Upon reaching the biofilm, the acidic microenvironment triggered nanoparticle degradation, releasing DNase to disrupt eDNA and vancomycin to target dispersed bacterial cells. This approach reduced S. aureus biofilm biomass by 70% compared to free drug combinations.

Liposomal systems provide another effective platform for enzyme and antimicrobial co-delivery. Liposomes composed of phosphatidylcholine and cholesterol can encapsulate hydrophilic enzymes like proteinase K in their aqueous core while loading hydrophobic antimicrobials like ciprofloxacin within the lipid bilayer. In Pseudomonas aeruginosa biofilms, proteinase K-loaded liposomes degraded biofilm proteins, reducing EPS viscosity and enabling deeper ciprofloxacin penetration. Studies showed a 2.5-fold increase in ciprofloxacin accumulation within the biofilm when delivered with proteinase K-loaded liposomes compared to free ciprofloxacin. This combination achieved a 90% reduction in viable P. aeruginosa cells within 24 hours.

Inorganic nanoparticles, such as mesoporous silica, offer high loading capacities and tunable surface properties for biofilm targeting. Silica nanoparticles functionalized with positively charged amines can adhere to negatively charged biofilm surfaces, enhancing local enzyme and antimicrobial concentrations. For example, DNase and tobramycin co-loaded silica nanoparticles effectively penetrated S. epidermidis biofilms, with DNase activity remaining stable for up to 72 hours. The nanoparticles reduced biofilm metabolic activity by 85% and prevented biofilm regrowth over 7 days.

The sequential release of enzymes and antimicrobials is critical for optimal biofilm disruption. Nanocarriers can be engineered to release DNase or proteinase K first, degrading the EPS and exposing bacterial cells to subsequent antimicrobial release. pH-responsive nanoparticles exploit the acidic biofilm microenvironment to trigger enzyme release, followed by sustained antimicrobial elution. In one study, proteinase K released from pH-responsive nanoparticles degraded P. aeruginosa EPS within 2 hours, enabling rapid rifampicin diffusion and killing 99% of biofilm bacteria within 12 hours.

Enzyme stability within nanocarriers is influenced by encapsulation methods. For instance, lyophilized DNase encapsulated in trehalose-containing nanoparticles retained full activity after 4 weeks of storage, whereas free DNase lost 60% activity under the same conditions. Similarly, proteinase K encapsulated in alginate-chitosan nanoparticles showed no activity loss after exposure to gastric enzymes, highlighting the protective effect of nanocarriers in harsh environments.

Targeted delivery to biofilms can be further enhanced by surface modifications. Nanoparticles conjugated with biofilm-specific peptides or antibodies exhibit higher binding affinity to EPS components. For example, nanoparticles functionalized with a peptide targeting S. aureus polysaccharide intercellular adhesin (PIA) showed 3-fold greater accumulation in biofilms compared to unmodified nanoparticles. This targeting strategy, combined with co-loaded DNase and daptomycin, reduced S. aureus biofilm viability by 95%.

Despite these advances, challenges remain in scaling up nanocarrier systems for clinical use. Batch-to-batch variability in nanoparticle synthesis, enzyme loading efficiency, and long-term stability must be addressed. Additionally, the potential for nanoparticle-induced immune responses requires careful evaluation. However, the ability of nanocarriers to protect enzymes, enhance biofilm penetration, and synergize with antimicrobials positions them as a transformative approach for treating persistent biofilm infections.

In conclusion, nanoparticle systems co-loaded with DNase, proteinase K, and antimicrobials represent a sophisticated strategy to overcome biofilm resistance. By leveraging the protective and penetrative properties of nanocarriers, these systems effectively disrupt EPS barriers and improve antimicrobial efficacy. Examples targeting staphylococcal and pseudomonal biofilms demonstrate the potential of this approach, offering a pathway to more effective treatments for chronic infections. Future developments in nanocarrier design and targeting will further optimize biofilm eradication while minimizing off-target effects.