Nanoparticles (NPs) are particles with more than one dimension between 1 and 100 nm. Because of their small size, they typically display different physical and chemical properties than the corresponding bulk materials. NPs have been used in many different applications, such as in electronics, optics, catalysis, and in biomedicine. Due to their colloidal nature, NPs are often immobilized on a solid substrate, such as glass or polymer-based materials, including biopolymers. Nanocellulose is a biopolymerbased nanomaterial that can be obtained from plants or bacterial biofilms. They can be processed into thin and highly hydrated films with high mechanical strength and can serve as a versatile substrate for NPs. Bacterial cellulose (BC) is also an interesting material for generating wound dressings. The combination of NPs and BC results in soft and flexible nanocomposites (BC-NPs) that can demonstrate novel properties and improve the functionality of wound dressings. 

BC-NP nanocomposites have previously been obtained by impregnating BC with the reactants needed for synthesis of the NPs and allowing the reaction to proceed in situ, inside and on the surface of the BC. This strategy limits the possibilities to control NP geometry and NP concentration and make synthesis of nanocomposites with more sophisticated compositions very challenging. In addition, the synthesis conditions used can potentially have negative effects on the properties of BC. 

The work presented in this thesis shows the possibility to produce well-defined, tunable BC-NP nanocomposites using self-assembly under very benign conditions that enable functionalization of BC with a wide range of different types of NPs. In addition to exploring the self-assembly process and the physical properties of these new BC-NP composites, several different applications were investigated. The functionalization of BC with gold nanoparticles (AuNPs) of different sizes and geometries was demonstrated. The resulting materials were used for development of a new sensor transduction technology, exploiting the optical response upon mechanical compression to detect biomolecules. BC-AuNP nanocomposites were also developed for monitoring of protease activity of wound pathogens, for catalysis, and for fabrication of ultra-black materials with unique absorption and scattering profiles of light in the visible and near infrared spectral range. In addition, the self-assembly process could be adopted for generating BC-mesoporous silica nanoparticles (MSNs) nanocomposite wound dressings. The resulting high surface area materials could be used as carriers for pH sensitive dyes. The pH-responsive BC-MSNs demonstrated adequate biocompatibility and allowed for monitoring of wound pH and for assessment of wound status. 

The strategies for functionalization of BC with inorganic NPs that was developed and explored in this thesis are highly versatile and allow for fabrication of a wide range of multifunctional nanocomposite materials. 

Hard-to-heal wounds cause significant patient suffering and place a heavy burden on healthcare systems, accounting for about 4 % of the entire healthcare budget globally. In hospital settings, up to 40% of beds are occupied by patients with wounds, and substantial resources are allocated to outpatient care. Wounds are highly susceptible to bacterial colonization, which can lead to infection and further delay healing. With the rise of multidrug-resistant bacteria, this challenge has become a global healthcare concern.

This thesis explores new antimicrobial strategies and techniques to modify nanocellulose wound dressings to enable both detection and treatment of infections in hard-to-heal wounds. Nanocellulose, and in particular bacterial nano-cellulose (BC), is an attractive wound dressing material that provides a moist wound microenvironment and a protective barrier against external pathogens but lacks inherent antimicrobial properties. Antimicrobial peptides (AMPs) have recently gained attention for wound treatment due to their potent antimicrobial effects and low risk of antibiotic resistance.

This thesis explores the possibility to functionalize BC wound dressings with AMPs, focusing on: i) enhancing AMPs loading, ii) preventing uncontrolled release, and iii) enabling triggered AMPs release to develop stimuli-responsive wound dressings. Initially, the physical adsorption of the AMPs PLNC8 αβ in BC was investigated. However, this approach resulted in low loading efficiency and uncontrolled release. To improve AMPs loading, mesoporous silica nanoparticles (MSNs) were self-assembled in the BC dressings, resulting in a BC-MSN composite with a threefold increase in specific surface area. The nanocomposites retained attractive wound dressing properties and AMPs loading proved to be more efficient, achieving up to a fourfold increase in AMPs concentrations. In a second approach loading of AMPs micelles and AMPs-loaded MSNs in a hyaluronic acid hydrogel that was subsequently grafted to BC was developed. This strategy ensured efficient AMPs loading and high antimicrobial activity while stimulating healing.

To attain a more precise AMPs release control, a strategy to coat AMPs-loaded MSNs with proteins was developed. Protein capped MSNs retained the AMPs in absence of proteases, while protease-triggered degradation of the capping led to a ~85% AMPs release. Protease activity is typically highly upregulated in infected wounds and the protease responsive AMPs release can thus facilitate efficient infection control. In addition to controlled AMPs delivery, this thesis explores novel AMPs designs. Starting with PLNC8 β, iterations of truncations, amino acid substitutions, and lipidation, lead to the discovery of a new class of sequence-optimized antimicrobial peptides (SOAPs) with broad-spectrum activity and potent antimicrobial action in the micromolar concentration range. To further enhance their efficacy and reduce the required dosage, SOAP peptides were loaded onto self-assembled BC-silver nanoparticle (AgNP) composite dressings, allowing for the simultaneous delivery of AMPs and silver ions.

Beyond infection treatment, this work also explores the potential of BC-MSN composite materials for wound infection detection. The high surface area of the composite was utilized to immobilize a pH-responsive molecule capable of detecting pH changes in the wound microenvironment indicative of infection. The immediate color change of the dressings in infected wounds could enable early and non-invasive wound status monitoring.

Overall, the functionalization strategies presented in this thesis provide a highly adaptable platform for development of multifunctional antimicrobial nano-cellulose wound dressings, offering new possibilities for advanced wound care.