Zhao Yuliang: Progress of smart nanomachines used in the treatment of major diseases

[Editor's note] 2020 November 29, the National Natural Science Fund Committee will cross the high-end scientific academic forum held in Beijing.

Li Suping1,2 Lu Zefang1,2 Nie Guangjun1,2 Zhao Yuliang1,2*

1.

2.

Zhao Yuliang Chinese Academy of Sciences academician , developing countries Sciences academician , director of the National Center for Nanoscience.

Li Suping is a researcher and doctoral supervisor of the National Nanoscience Center.

Summary

The incidence and fatality rate of major diseases in my country remain high.

Keywords: smart nanomachine; drug delivery; precision medicine; clinical translation; interdisciplinary science

1 The strategic significance of medical nanotechnology

In recent years, the level of medical care in my country has been continuously improved, and the life expectancy of the population has been continuously extended.

In the development of new drugs, in addition to fierce competition in new mechanisms, new targets and new drug molecules, drug delivery runs through the development of new drugs and has become the hotspot and frontier of competition among major pharmaceutical companies in the world [2].

Nano-carriers and/or nano-drugs, as an important form of innovative pharmaceutical preparations, have their own unique organ targeting and circulation properties.

The early clinical practice of nanomedicine and the emerging research of intelligent nanomachines both show that the precise assembly of nanomedicine, intelligent response, biological effects in vivo, biosafety, etc.

2 The third generation of nanomedicine-the design principles and development trends of medical intelligent nanomachines

Nanotechnology has widely penetrated into all aspects of disease prevention, detection, imaging, and treatment.

However, due to the complexity of the organism, it is a greater challenge for the drug to reach the lesion site and exert the expected effect.

Using smart nanorobots to treat diseases is a long-standing wish of mankind.

Figure 1 The design concept of an intelligent nanorobot

Due to the complexity and prospective nature of nanorobots, there have not been reports of successful application of nanorobots in clinical trials.

Molecular self-assembly focuses more on the basis of materials science and chemistry.
According to internal and external stimuli related to diseases and treatments, one-to-one correspondence of functional modules, such as sensitive bonds that can be broken, charge reversal, and detachable/degradable shells, can be used for nanomachines.
Time and space control (Figure 2a).
The polymer molecule has a large number of reactive groups that can be reacted, and small molecule drugs can be connected before and after monomer polymerization, and sensitive and targeting modules can be introduced to achieve focus-specific enrichment, pH response, glutathione response, and reactive oxygen species.
The functions of response, temperature response, and hypoxia response are used to form nanostructures by adjusting the ratio of hydrophilicity to hydrophobicity (Figure 2b).
Similar to polymers, peptides also introduce functional modules during molecular synthesis, forming nanospheres or nanofibers through hydrophilic-hydrophobic interactions/hydrogen bonds, and protect drugs from plasma protein interference in the blood circulation (Figure 2c).
The difference is that the peptide sequence itself can perform targeting, acid response, enzyme response and pharmacological functions, and the synthesis and modification process is more mature, so it is more suitable for large-scale production.
In addition to molecular-level self-assembly, the secondary assembly and disassembly of nanoparticles under mechanical, chemical and optical stimulation to achieve particle size changes at thrombus or tumor sites is of great significance for increasing drug retention in the lesion area (Figure 2d) .
The emergence of DNA origami nanocarriers incorporates the increased level of disease-related protein markers into stimulus factors.
After partially complementary paired nucleic acid aptamers bind to their target proteins or small molecules, they dissociate from the complementary strands to open the molecular lock, causing origami Conformation changes, exposure or release of internal drugs; by precisely controlling the number and position of single strands and molecular locks of nucleic acid aptamers, the concentration limits of multiple markers and the logical relationship between responses can be designed to more accurately identify target cells; Loading specific nucleic acid sequences inside and performing chain replacement or chain cross-linking with molecular locks can realize positive and negative feedback of drug release, and the release is controllable and reversible, which is closer to the concept of nanomachines.
At present, the in vivo application of DNA nanomachines has been preliminarily verified.
The nucleolin receptor specifically up-regulated on the surface of tumor vascular endothelium serves as a target and stimulation to control DNA nanomachines to expose thrombin to block tumor blood vessels (Figure 2e) [8].
However, DNA nanomachines need to be further improved in terms of increasing the stability of nucleic acids in vivo, broadening the library of nucleic acid aptamers, reducing synthesis costs, and designing all-in-one nanomachines with complete logic circuits.
Finally, for the types of nanomachines that can respond to external stimuli, such as light, sound, and magnetic signals, to report the position or state of the nanomachine, its special significance is that the local nanomachine changes or responds to the lesion under the guidance of the image, releasing or exposing the contents, or Use the properties of nanomaterials to produce sound, heat and free radical killing, while monitoring the treatment Process, visualize the distribution and metabolic behavior of nanomachines in the body, and provide a more precise treatment window for connecting follow-up therapies.
However, such nanomachines need to comprehensively consider the type of external stimulus, the depth of access, the resolution, and the short-term and long-term toxicity of nanomaterials that can respond to these stimuli.
There are high challenges in selecting the type of disease.

Figure 2 Application of bottom-up molecular self-assembly nanomachines in disease treatment

(A) Disease microenvironment and stimulus response elements in organisms[12]; (b) Secondary targeting polymer micellar nanomachines for tumor photodynamic therapy[13]; (c) Anti-tumor angiogenesis Therapeutic polypeptide self-assembled nanomachine[14]; (d) Particle secondary assembly nanomachine for thrombolytic therapy[15]; (e) DNA nanomachine for tumor vascular embolization therapy[8]

The advantage of bottom-up self-assembled nanomachines is to clarify the difficulties in the delivery process and carry out targeted design; with the further improvement of understanding of disease pathology, modular nanomachines can quickly adjust synthesis strategies and are expected to develop into platform technologies.
However, this strategy of one-to-one correspondence between problems and solutions also limits the efficiency of drug delivery; that is, since the targeting or response efficiency of each module cannot reach 100%, the more modules are introduced, the possibility of performing functions according to the design.
The lower the sex.
On the one hand, it is necessary to strengthen the basic research on the structure-activity relationship of nanostructures to achieve breakthroughs on the key issues of limiting delivery efficiency; on the other hand, the design of nanomachines needs to learn from all aspects of the cell life process.
At the molecular level, natural nanomachines have motor proteins that transport substances on microfilaments and microtubules, and at the subcellular level there are ribosomes that translate proteins and extracellular vesicles that transport chemical mediators, except in molecular biology.
Understanding the working principles of natural nanomachines to help artificial design, and directly using natural nanostructures to deliver drugs is also a major development direction.
Natural proteins can be expressed in a eukaryotic/prokaryotic system for large-scale production, and their hydrophobic cavities can be used to load small molecule drugs (Figure 3a).
Due to the presence of these proteins in the body, the possible immunogenicity is low.
At the same time, it uses the interaction between the protein and its ligand and the protein allosteric caused by pH and ATP to achieve targeting and response functions.
It is the smallest medical nanorobot [ 16, 17].
Protein nanorobots are difficult to encapsulate macromolecular drugs due to the limitation of the cavity size.
Exosomes exuded by endosomes and microparticles shed from the plasma membrane can carry drugs through electroporation or genetic engineering.
Since extracellular vesicles carry cellular biological information and mediate interactions with specific cell types, they are also a representative type of good natural intelligent nanomachines (Figure 3b).
At present, many large pharmaceutical companies have paid attention to the potential of extracellular vesicles to deliver nucleic acid drugs.
In June 2020 alone, more than $1 billion in exosomal drug carrier related transactions occurred [18].
In addition, cell and bacterial membranes are closely related to their physiological effects.
The membrane structure is extracted and synthetic nanoparticles are used as support to play the role of drug delivery, immune stimulation and toxin removal, providing new ideas for nanomachines to simulate cell functions (Figure 3c).

Figure 3 Natural drug-loaded nanorobots from top to bottom

(a) ATP-responsive natural protein nanomachines deliver chemotherapeutic drugs for tumor treatment [17]; (b) extracellular vesicle nanomachines represented by exosomes are used for drug delivery; (c) platelet membrane-encapsulated nanomachines Eliminate pathogens [4]

During the preparation of vesicles or membrane-coated nanomachines, changes in phospholipids and protein components may reduce drug delivery efficiency and pharmacological effects; on the other hand, such nanomachines often lack response modules and cannot achieve controlled drug release.
Therefore, combining the bottom-up and top-down strategies can maximize their respective advantages.
A simple strategy is to fuse stimulus-responsive phospholipids with natural membrane structures to assemble nanomachines, fully integrating their respective response and targeting advantages; another relatively complex strategy is to load nanomachines on the surface of cells or bacteria.
The current specific enrichment of nanocarriers still relies on passive capture, that is, nanocarriers stay in the focus area through size effect and protein interaction.
A few studies use enzymatic, chemical and ultrasonic cavitation to generate bubbles to promote the increase of nanocarriers.
The depth of penetration at the lesion site, but these strategies have problems such as uncontrollable movement direction and short duration of movement.
Even if the magnetic field is used to control the directional movement of the nanocarrier, it is limited by the accuracy of the magnetic field.
Therefore, nanomachines are not yet able to actively enrich the lesion area by using the concentration gradient of tropic factors like cells.
Nanoparticles are loaded on the surface of cells or bacteria.
Cells/bacteria provide targeting and power.
Nanoparticles provide drug loading and sensitive response.
For example, magnetotactic bacteria move to hypoxic areas along the magnetic line of induction and oxygen gradient.
Tumor core [19], T cell surface nanoparticles respond to the increase of cell surface sulfhydryl levels caused by T cell receptor activation, and release drugs to enhance or suppress immunity [20, 21].
This type of research is currently the fourth of the current therapeutic nanorobots.
The big function provides an important reference.
However, this strategy also faces the impact of loading nanoparticles on cells/bacteria, the penetration of micron-sized cells/bacteria in tissues, early endocytosis of nanoparticles, and immunogenicity.
In the future, as the understanding of materials science, biology, physiology and pathology is further deepened, the design of medical nanorobots by simulating or using natural sensors and effectors can realize the unification of top-down and bottom-up.

3 Key challenges and bottlenecks in the research of smart nanomachine drugs

Intelligent nanomachines are the product of multi-disciplinary intersections.
There are not only a large number of multi-disciplinary basic research problems, but also huge clinical demand-oriented technical challenges.
Therefore, from both basic and application aspects, the research of smart nanomachines needs to break through several important evaluations and challenges.

3.
1 Key scientific issues in the basic research of medical nanomachines

Although there are various smart nanomachine designs, the research on the biological effects and structure-activity relationship of nanomachines is still not deep enough.
Safety is the prerequisite for the use of medicines, so the biological effects of medical nanomachines, especially toxicological research, are the basis for the application of medical nanomaterials.
At the level of health and disease animal models, use a variety of methods to mark and detect the absorption, distribution, metabolism and excretion of nanomachines and their degradation products, and to map the time and space distribution of nanomachines from entering to exiting the living body in detail; at the tissue level, in addition to traditional Pathological slices, using emerging technologies such as mass spectrometry and single-cell multi-omics to describe the distribution of cell types and the signal pathways affected in detail; at the cellular level, fully study the endocytic pathways of nanomachines and the pathways to inflammation, programmed death, and The influence of functional regulation pathways.
The in-depth study of this series of issues is not only a necessary part of the preclinical research of medical nanomachines, but also an important method for evaluating the biological safety of nanomaterials in the environment and commodities.

The characterization methods of in vivo and in vitro nanomachines are not perfect, that is, the physical and chemical properties of nanomachines, including material, particle size, charge, shape, specific surface area, hardness, deformability, surface ligand modification type and density, are related to the protein crown on the particle surface.
Formation, the removal of mononuclear phagocyte system, the distribution and metabolism of the body, the enrichment and retention of the lesion area, and the influence of the endocytosis of target cells.
These unknown problems hinder the horizontal comparison between different construction strategies and the development of nanomachines.
Dig further.
Therefore, it is of great significance to gradually establish a set of evaluation systems for the structure-efficiency relationship of medical nanomachines.
On this basis, building a database, computer learning and predicting the delivery efficiency of newly designed nanomachines, can greatly promote the development process of intelligent nanomachines.

3.
2 Key technical challenges in the application research of smart nanomachines

The application research of smart nanomachines is to use existing materials and biotechnology to design sophisticated, practical and ingenious nanocarriers based on specific needs in clinical practice, combining drug action sites and drug delivery methods.
The momentum, heat and mass transfer of the synthesis of industrial production and laboratory small systems are different, and there will be unknown risks in the process of expanding the system; at the same time, clinical applications put forward higher requirements for the uniformity and batch-to-batch stability of nanomedicine Therefore, the design of nanomachines needs to balance functionality and complexity.
Screening more suitable indications and patients is a key issue in the transformation of smart nanomachines.

4 Some thoughts on accelerating the development of smart nanomedicine

At present, more than 50% of the research results related to nano-drug delivery published in the field of nano-medicine belong to the field of anti-tumor therapy.
This may be due to the extensiveness and severity of tumors, the huge market of anti-tumor drugs, the wide application of anti-tumor nano-drugs in clinical practice, people’s deep understanding of tumor cells and their microenvironment, and the relatively mature biology of anti-tumor nano-drugs.
Evaluation System.
However, nanomedicine faces multiple speed-limiting steps from entering blood vessels to being engulfed by cells in the tumor microenvironment, requiring the design and construction of multifunctional nanomachines.
Relatively speaking, diseases suitable for treatment with nanomachines should have the following characteristics:

(1) Compared with standard clinical treatment programs, nano-delivery can increase the concentration of drugs in the lesion area or change the way of administration to reduce patient suffering and increase compliance;

(2) The site of drug effect should be easy to reach.
For example, for intravenous infusion of nanomachines, intravascular drugs are better than extracellular effects than intracellular effects;

(3) Compared with the surrounding normal tissues, the lesion area has more obvious changes in biochemical properties;

(4) There are potential treatment options but lack of delivery vehicles for in vivo applications.
The methods of administration for these diseases are not all intravenous infusions; therefore, it is possible to study the special barriers for nanomedicine to reach the lesion area through nasal, oral, and eye drops, and closely combine with clinical needs to clarify the indications and explore the feasibility of treatment.
Broaden the scope of application of nano-medicine and accelerate clinical transformation.

Precise drug delivery is a major trend in patient medication.
Detecting the markers in the patient's body before treatment is essential for choosing the appropriate treatment plan.
Since there are many factors that affect the delivery of nanomachines in vivo, the supporting detection methods of nanomachines cannot only use biomarkers as indicators.
One idea is to design an empty carrier that has the same structure as the drug-loaded nanomachine and carries the reporter molecule.
When the nanomachine is turned on, the reporter molecule activates and sends a signal to visualize the operation of the nanomachine in the body.
According to this, the patient can be classified, which may increase the clinical use of nanomedicine.
The success rate of the test.

5 Several suggestions to promote the development of medical nanotechnology industry

Smart nanomachine medicine is a highly intersecting multidisciplinary field.
Combining the internal motivations of clinical practice and interdisciplinary development, promote the better combination of basic research and clinical application of medical nanotechnology, and form my country's key core technology system in the field of medicine and health in the future.

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