Implanted synthetic cells trigger tissue angiogenesis through de novo production of recombinant growth factors.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106 protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane-like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Nanoparticles target early-stage breast cancer metastasis in vivo.

Despite advances in cancer therapy, treating cancer after it has metastasized remains an unmet clinical challenge. In this study we demonstrate that 100 nm liposomes target triple-negative murine breast-cancer metastases post intravenous administration. Metastatic breast cancer was induced in BALB/c mice either experimentally, by a tail vein injection of 4T1 cells, or spontaneously, after implanting a primary tumor xenograft. To track their biodistribution in vivo the liposomes were labeled with multi-modal diagnostic agents, including indocyanine green and rhodamine for whole-animal fluorescent imaging, gadolinium for magnetic resonance imaging (MRI), and europium for a quantitative biodistribution analysis. The accumulation of liposomes in the metastases peaked at 24 h post the intravenous administration, similar to the time they peaked in the primary tumor. The efficiency of liposomal targeting to the metastatic tissue exceeded that of a non-liposomal agent by 4.5-fold. Liposomes were detected at very early stages in the metastatic progression, including metastatic lesions smaller than 2 mm in diameter. Surprisingly, while nanoparticles target breast cancer metastasis, they may also be found in elevated levels in the pre-metastatic niche, several days before metastases are visualized by MRI or histologically in the tissue. This study highlights the promise of diagnostic and therapeutic nanoparticles for treating metastatic cancer, possibly even for preventing the onset of the metastatic dissemination by targeting the pre-metastatic niche.

Chemical Zymogens and Transmembrane Activation of Transcription in Synthetic Cells.

In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

A mucoadhesive patch loaded with freeze-dried liposomes for the local treatment of oral tumors.

Oral cancers affect millions of people globally, with increasing incidences among adults aged 35 and above. Poor drug uptake by lesions in the oral cavity following systemic administration, as well as limited localized treatment modalities for oral tumors, result in poor patient quality of life and high mortality. Here, we describe a solid, dissolvable, bioadhesive alginate patch containing freeze-dried doxorubicin-loaded liposomes as a local treatment for oral tumors located on the tongue. By varying the alginate-to-liposome ratio in the mucoadhesive patch, we could control the degree of bioadhesion to the tongue and the release profile of the drug-loaded liposomes from the matrix. In vitro, exposing squamous cell carcinoma (SCC) to the alginate mucoadhesive patch or tablet resulted in dose-dependent cancer-cell death. In vivo, the efficacy of the local treatment was demonstrated in mice bearing orthotopic SCC tumors in the tongue. The bioadhesive patch, applied directly above the lesion, significantly reduced the tumor size and treatment-associated side effects compared to implanted patches or systemic drug administration. This study demonstrates that local bioadhesive therapies are effective in treating cancers of the oral cavity.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Nanoparticles targeting the infarcted heart.

We report a nanoparticulate system capable of targeting the heart after myocardial infarction (MI). Targeting is based on overexpression of angiotensin II type 1 (AT1) receptor in the infarcted heart. Liposomes 142 nm in diameter were conjugated with a ligand specific to AT1. The nanoparticles were able to specifically target cardiac cells in vitro, and in the infarcted heart after intravenous injection in vivo. This system may be useful for delivering therapeutic agents specifically to the infarcted heart.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.

Lung targeted liposomes for treating ARDS.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1ß cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

Interactions between adsorbed hydrogenated soy phosphatidylcholine (HSPC) vesicles at physiologically high pressures and salt concentrations.

Using a surface force balance, we measured normal and shear interactions as a function of surface separation between layers of hydrogenated soy phosphatidylcholine (HSPC) small unilamellar vesicles (SUVs) adsorbed from dispersion at physiologically high salt concentrations (0.15 M NaNO3). Cryo-scanning electron microscopy shows that each surface is coated by a close-packed HSPC-SUV layer with an overlayer of liposomes on top. A clear attractive interaction between the liposome layers is seen upon approach and separation, followed by a steric repulsion upon further compression. The shear forces reveal low friction coefficients (µ = 0.008-0.0006) up to contact pressures of at least 6 MPa, comparable to those observed in the major joints. The spread in µ-values may be qualitatively accounted for by different local liposome structure at different contact points, suggesting that the intrinsic friction of the HSPC-SUV layers at this salt concentration is closer to the lower limit (µ = ~0.0006). This low friction is attributed to the hydration lubrication mechanism arising from rubbing of the hydrated phosphocholine-headgroup layers exposed at the outer surface of each liposome, and provides support for the conjecture that phospholipids may play a significant role in biological lubrication.

Robotics, microfluidics, nanotechnology and AI in the synthesis and evaluation of liposomes and polymeric drug delivery systems.

The field of nanotechnology and personalised medicine is undergoing drastic changes in the approach and efficiency of experimentation. The COVID-19 pandemic has spiralled into mass stagnation of major laboratories around the globe and led to increased investment into remote systems for nanoparticle experiments. A significant number of laboratories now operate using automated systems; however, the extension to nanoparticle preparation and artificial intelligence-dependent databases holds great translational promise. The strive to combine automation with artificial intelligence (AI) grants the ability to optimise targeted therapeutic nanoparticles for unique cell types and patients. In this perspective, the current and future trends of automated approaches to nanomedicine synthesis are discussed and compared with traditional methods.

Liposomes act as effective biolubricants for friction reduction in human synovial joints.

Phospholipids (PL) form the matrix of biological membranes and of the lipoprotein envelope monolayer, and are responsible for many of the unique physicochemical, biochemical, and biological properties of these supermolecular bioassemblies. It was suggested that phospholipids present in the synovial fluid (SF) and on the surface of articular cartilage have major involvement in the low friction of cartilage, which is essential for proper mobility of synovial joints. In pathologies, such as impaired biolubrication (leading to common joint disorders such as osteoarthritis), the level of phospholipids in the SF is reduced. Using a human-sourced cartilage-on-cartilage setup, we studied to what extent and how phospholipids act as highly effective cartilage biolubricants. We found that large multilamellar vesicles (MLV), >800 nm in diameter, composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or of a mixture of DMPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are superior lubricants in comparison to MLV composed of other phosphatidylcholines. Introducing cholesterol into liposomes resulted in less effective lubricants. DMPC-MLV was also superior to small unilamellar vesicles (SUV), <100 nm in diameter, composed of DMPC. MLV are superior to SUV due to MLV retention at and near (<200 microm below) the cartilage surface, while SUV penetrate deeper into the cartilage (450-730 microm). Superiority of specific PL compositions is explained by the thermotropic behavior (including compressibility) of the lipid bilayer. Correlating physicochemical properties of the MLV with the friction results suggests that MLV having lipid bilayers in the liquid-disordered phase and having a solid-ordered to liquid-disordered phase transition temperature slightly below physiological temperature are optimal for lubrication. High phospholipid headgroup hydration, high compressibility, and softness are the common denominators of all efficient PL compositions. The high efficiency of DMPC-MLV and DMPC/DPPC-MLV as cartilage lubricants combined with their resistance to degradation at 37 degrees C supports further evaluation of these MLV for treatment of joint impairments related to poor lubrication. This work also demonstrates the relevance of basic physicochemical properties of phospholipids to their activities in biological systems.

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

Mucoadhesive Hybrid Polymer/Liposome Pastes Based on Modified Polysaccharides.

Mucoadhesive hybrid polymer/liposome paste is a new drug delivery system presenting controllable and tailorable delivery mechanism. By using mucoadhesive material, the delivery can be more specific and local. Here, we present a study investigating the effect of polymer type, concentration, functional end group, and cross-linking on the release profile of nanoliposomes from polymer pastes. Polymer pastes can be expected to combine the mucoadhesion mechanisms of dry and wet dosage forms but have not been studied extensively. To better understand the mucoadhesion of pastes, we investigated a series of pastes based on the same polymer and used different chemical modifications that can produce interactions at different levels. Native and thiolated polymers presented enhanced mucoadhesion in a wet environment in comparison to acrylated polymers which dissolved rapidly because of the enhanced solubility of PEG chains in water. Paste cross-linking resulted in a sustained release profile compared to non-cross-linked pastes. Pectin-SH pastes, especially 3% (w/v), showed a linear liposomal release profile which is ascribed to the combination of ionic cross-linking and disulfide bridging. By configuring the polymer type or concentration, we can control the release mechanisms and achieve distinct inherent properties which can be applied for diverse medical applications.

Sodium bicarbonate nanoparticles modulate the tumor pH and enhance the cellular uptake of doxorubicin.

Acidic pH in the tumor microenvironment is associated with cancer metabolism and creates a physiological barrier that prevents from drugs to penetrate cells. Specifically, ionizable weak-base drugs, such as doxorubicin, freely permeate membranes in their uncharged form, however, in the acidic tumor microenvironment these drugs become charged and their cellular permeability is retarded. In this study, 100-nm liposomes loaded with sodium bicarbonate were used as adjuvants to elevate the tumor pH. Combined treatment of triple-negative breast cancer cells (4T1) with doxorubicin and sodium-bicarbonate enhanced drug uptake and increased its anti-cancer activity. In vivo, mice bearing orthotropic 4T1 breast cancer tumors were administered either liposomal or free bicarbonate intravenously. 3.7 ±â€¯0.3% of the injected liposomal dose was detected in the tumor after twenty-four hours, compared to 0.17% ±â€¯0.04% in the group injected free non-liposomal bicarbonate, a 21-fold increase. Analyzing nanoparticle biodistribution within the tumor tissue revealed that 93% of the PEGylated liposomes accumulated in the extracellular matrix, while 7% were detected intracellularly. Mice administered bicarbonate-loaded liposomes reached an intra-tumor pH value of 7.38 ±â€¯0.04. Treating tumors with liposomal bicarbonate combined with a sub-therapeutic dose of doxorubicin achieved an improved therapeutic outcome, compared to mice treated with doxorubicin or bicarbonate alone. Interestingly, analysis of the tumor microenvironment demonstrated an increase in immune cell' population (T-cell, B-cell and macrophages) in tumors treated with liposomal bicarbonate. This study demonstrates that targeting metabolic adjuvants with nanoparticles to the tumor microenvironment can enhance anticancer drug activity and improve treatment.

Collagenase Nanoparticles Enhance the Penetration of Drugs into Pancreatic Tumors.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Therapeutic nanoparticles penetrate leaves and deliver nutrients to agricultural crops.

As the world population grows, there is a need for efficient agricultural technologies to provide global food requirements and reduce environmental toll. In medicine, nanoscale drug delivery systems grant improved therapeutic precision by overcoming biological barriers and enhancing drug targeting to diseased tissues. Here, we loaded nanoscale drug-delivery systems with agricultural nutrients, and applied them to the leaves of tomato plants. We show that the nanoparticles - liposomes composed of plant-derived lipids, penetrate the leaf and translocate in a bidirectional manner, distributing to other leaves and to the roots. The liposomes were then internalized by the plant cells, where they released their active ingredient. Up to 33% of the applied nanoparticles penetrated the leaf, compared to less than one percent of free-molecules applied in a similar manner. In our study, tomato plants treated with liposomes loaded with Fe and Mg overcame acute nutrient deficiency which was not treatable using ordinary agricultural nutrients. Furthermore, to address regulatory concerns regarding airborne nanoparticles, we rationally designed liposomes that were stable only over short spraying distances (less than 2 meters), while the liposomes disintegrated into safe molecular building blocks (phospholipids) over longer airborne distances. These findings support expanding the implementation of nanotechnology for delivering micronutrients to agricultural crops for increasing yield.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

Mucoadhesive alginate pastes with embedded liposomes for local oral drug delivery.

Oral cancers are extremely common among adults with increasing incidences due to human papillomavirus, while treatment modalities are limited. This study aims to develop a new oral mucoadhesive delivery system based on the combination of alginate and liposomes. The polymer provides adhesion properties and induces local release of the drug-loaded carriers, while the liposomes protect the drug from degradation and improve its absorption into the cells. Three hybrid alginate/liposomes delivery systems were investigated: a hybrid paste, which presented excellent adhesive capabilities, yet fast burst release of 90% after 2h; a hybrid hydrogel, demonstrating controllable release rates of 5%, 30% or 60% after 2h but poor mucoadhesive properties. These findings led to the development of a hybrid cross-linked paste. Polymer retention studies demonstrated that 80% of the crosslinked paste was retained on tongue tissue compared to 50% retention of the non-cross-linked pastes, verifying its superior mucoadhesion. The hybrid cross-linked paste presented controllable release rate of 20% after 2h. Alginate paste incorporating doxorubicin loaded liposomes presented similar release rates and were highly effective in promoting cancer cell death. Thus, our innovative formulation, including both desired characteristics of mucoadhesion and sustained liposomes release, is an important milestone in the development of a new potential treatment for oral cancer.

Proteolytic Nanoparticles Replace a Surgical Blade by Controllably Remodeling the Oral Connective Tissue.

Surgical blades are common medical tools. However, blades cannot distinguish between healthy and diseased tissue, thereby creating unnecessary damage, lengthening recovery, and increasing pain. We propose that surgical procedures can rely on natural tissue remodeling tools-enzymes, which are the same tools our body uses to repair itself. Through a combination of nanotechnology and a controllably activated proteolytic enzyme, we performed a targeted surgical task in the oral cavity. More specifically, we engineered nanoparticles that contain collagenase in a deactivated form. Once placed at the surgical site, collagenase was released at a therapeutic concentration and activated by calcium, its biological cofactor that is naturally present in the tissue. Enhanced periodontal remodeling was recorded due to enzymatic cleavage of the supracrestal collagen fibers that connect the teeth to the underlying bone. When positioned in their new orientation, natural tissue repair mechanisms supported soft and hard tissue recovery and reduced tooth relapse. Through the combination of nanotechnology and proteolytic enzymes, localized surgical procedures can now be less invasive.