TWI848332B - Self-assembled nanoparticle and use thereof for anti-angiogenesis - Google Patents

Abstract

Provided is a nanoparticle or a pharmaceutical composition including the same for treating or remitting a neovascularization or an angiogenesis in eye segments, and the nanoparticle includes a hyaluronic acid and a therapeutic peptide. Also provided is a method for inhibiting formation or growth of blood vessels by administration of the nanoparticle or the pharmaceutical composition of the present disclosure to a subject in need thereof.

Description

Anti-angiogenic self-assembled nanoparticles and their uses

The present disclosure relates to self-assembled nanoparticles and their use in treating angiogenesis-related diseases or disorders, especially ocular angiogenesis-related diseases or disorders.

Eye diseases, such as age-related macular degeneration (AMD), are becoming more common around the world due to an aging population and excessive use of screen devices such as mobile phones and laptops, which causes eye fatigue and increases the risk of eye diseases. According to a 2021 World Health Organization (WHO) report, at least 220 million people worldwide suffer from visual impairment, of which about 200 million have moderate or severe visual impairment or blindness.

Feizi, S. et al. (Eye and Vision, 4(1): p.28, 2017) have shown that corneal angiogenesis plays an important role in visual impairment and blindness. According to statistics, approximately 1.4 million people experience corneal neovascularization (CoNV) each year, and 12% of them subsequently lose their vision. Corneal angiogenesis is also a common complication of corneal infection. The prevalence of infectious keratitis reflects the overall situation of corneal angiogenesis worldwide. Approximately 15% (6 million people) of blindness worldwide is caused by chlamydia infection; onchocerciasis infection is another major cause of blindness due to corneal angiogenesis. This type of infection has caused approximately 270,000 cases of blindness, and approximately 120 million people worldwide are at risk of infection. In the United States, it is estimated that there are approximately 500,000 cases of herpetic keratitis. In addition, long-term contact lens wear, especially soft hydrogel contact lenses, is also a factor causing corneal angiogenesis; it is estimated that 1.3% of the 9 million contact lens wearers have corneal neovascularization. In addition, the repair process when the cornea is chemically damaged (such as paint removers, dyes, acids and alkalis) can also lead to corneal angiogenesis. In the United States, the prevalence of corneal angiogenesis caused by various chemicals is about 37,000 people.

In addition to corneal angiogenesis, the formation and growth of new blood vessels in the posterior segment (e.g., choroidal neovascularization, ChNV) is also a major cause of serious eye disease and vision loss. This may occur in a variety of conditions, including individuals with degenerative macular degeneration (AMD), diabetic retinopathy (DR), and severe visual impairment.

Currently, the main treatments for eye diseases include oral steroids and intravitreal injections. However, long-term use of steroids can lead to side effects such as weight gain, high blood pressure, elevated blood sugar, glaucoma, cataracts, and gastrointestinal symptoms. Steroids also suppress the protective effects of the immune system, thereby increasing the risk of infections such as recurrence of herpes simplex. Intravitreal injections usually use anti-vascular endothelial growth factor (anti-VEGF) drugs, however, frequent injections of such drugs may lead to complications such as endophthalmitis, hemorrhage, high intraocular pressure, inflammation, cataracts, and retinal detachment.

Therefore, there is an urgent need for treatments that can effectively inhibit angiogenesis and deliver drugs in situ .

The present disclosure provides a self-assembled nanoparticle for inhibiting angiogenesis or new blood vessel formation. Such nanoparticles can be used, for example, to treat eye diseases and/or disorders.

Another aspect of the present disclosure is to provide a method for preparing the above-mentioned nanoparticles.

Another aspect of the present disclosure is to provide a composition comprising the nanoparticles of the present disclosure, such as a pharmaceutical composition.

Another aspect of the present disclosure is to provide a composition comprising the nanoparticles disclosed herein for treating or alleviating ocular angiogenesis, such as a pharmaceutical composition for alleviating ocular angiogenesis.

Another aspect of the present disclosure is to provide a method for treating or improving ocular angiogenesis-related diseases or conditions (such as degenerative macular degeneration (AMD)), which comprises administering the nanoparticles or compositions thereof disclosed herein to an individual in need thereof.

Another aspect of the present disclosure is to provide the use of the nanoparticles disclosed herein in the preparation of drugs.

To achieve the above aspects, the present disclosure provides a self-assembled nanoparticle comprising hyaluronic acid (HA) and a therapeutic peptide. In some embodiments, the self-assembled nanoparticles of the present disclosure are loaded with a given amount of therapeutic peptide at least 70% w/w, such as 70% w/w, 71% w/w, 72% w/w, 73% w/w, 74% w/w, 75% w/w, 76% w/w, 77% w/w, 78% w/w, 79% w/w, 80% w/w, 81% w/w, 82% w/ w, 83% w/w, 84% w/w, 85% w/w, 86% w/w, 87% w/w, 88% w/w, 89% w/w, 90% w/w, 91% w/w, 92% w/w, 93% w/w, 94% w/w, 95% w/w, 96% w/w, 97% w/w, 98% w/w, 99% w/w or higher amount of therapeutic peptide. In some specific embodiments, the self-assembled nanoparticles disclosed herein have a drug loading rate of about 70% to 99% w/w of therapeutic peptide, such as about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90% or about 95% to 99% of the given amount of therapeutic peptide.

In some embodiments, the size of the nanoparticles disclosed herein is about 100 to 400 nm, such as about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, or 350 to 400 nm. In some embodiments, the size of the nanoparticles disclosed herein is about 150 to 350 nm. In some embodiments, the polydispersity index of the nanoparticles disclosed herein is about 0.001 to 0.7, such as about 0.001 to 0.01, about 0.01 to 0.05, about 0.05 to 0.1, about 0.1 to 0.15, about 0.15 to 0.2, about 0.2 to 0.25, about 0.25 to 0.3, about 0.3 to 0.35, about 0.35 to 0.4, about 0.4 to 0.45, about 0.45 to 0.5, about 0.5 to 0.55, about 0.55 to 0.6, about 0.6 to 0.65, or about 0.65 to 0.7. In some specific embodiments, the zeta potential of the nanoparticles disclosed herein ranges from -40mV to 40mV, such as -40mV to -35mV, -35mV to -30mV, -30mV to -25mV, -25mV to -20mV, -20mV to -15mV, -15mV to -10mV, -10mV to -5mV, -5mV to 0mV, 0mV to 5mV, 5mV to 10mV, 10mV to 15mV, 15mV to 20mV, 20mV to 25mV, 25mV to 30mV, 30mV to 35mV, or 35mV to 40mV. In some specific embodiments, the zeta potential of the nanoparticles disclosed herein is about -35mV to 35mV.

In some specific embodiments, the nanoparticles disclosed herein contain therapeutic peptides, which exhibit antioxidant, anti-inflammatory and anti-angiogenic properties in vascular endothelial cells, especially the property of inhibiting the production of reactive oxygen species (ROS), thereby inhibiting the expression of vascular endothelial growth factor (VEGF). In some specific embodiments, the nanoparticles disclosed herein contain inhibitory peptides targeting Nox2, such as gp91 ds-tat peptides.

In some embodiments, the present disclosure provides a method for inhibiting blood vessel formation and/or growth, comprising administering the nanoparticles of the present disclosure to an individual in need thereof. In some embodiments, the blood vessel is an ocular blood vessel, wherein the nanoparticles are administered to the eye of the individual. In some embodiments, the eye includes an anterior segment and a posterior segment, wherein the anterior segment includes the cornea, conjunctiva, iris and lens; the posterior segment includes the anterior vitreous membrane, vitreous humor, retina, optic nerve, choroid and sclera. In some embodiments, the anterior segment refers to the cornea, and the posterior segment refers to the retina and choroid.

In some embodiments, the nanoparticles are substantially positively charged (zeta potential>0 mV) and are administered to the anterior segment of the eye of a subject in need thereof via an eye drop formulation. In some embodiments, the nanoparticles are substantially negatively charged (zeta potential<0 mV) and are administered to the posterior segment of the eye of a subject in need thereof via intravitreal injection. In some embodiments, the nanoparticles administered to the posterior segment of the eye pass through the retina and reach the choroid.

In some embodiments, the individual suffers from angiogenesis or angiogenesis-related disorder, such as solid tumors (e.g., cancer and malignant sarcomas), angiofibroma, arteriovenous malformations, atherosclerosis, hemangiomatosis, vascular adhesions, Maffucci syndrome, Osler-Weber-Rendu disease, inflammation, or abnormal wound healing.

In some embodiments, the subject has an ocular angiogenesis-related disorder, such as degenerative macular degeneration (AMD), retinal artery or vein occlusion, retinal branch vein occlusion, retinopathy of prematurity (ROP), neovascular glaucoma, corneal neovascularization (CoNV), diabetic macular edema (DME), acute idiopathic macular degeneration, polypoidal choroidal vasculopathy, ischemic diabetic retinopathy, pigmentary retinitis (RP), cone cell dystrophy, Behcet's disease, proliferative vitreoretinopathy (PVR), retinitis, uveitis, Leber's hereditary optic neuropathy (Leber's hereditary optic neuropathy). neuropathy), retinal detachment, retinal pigment epithelial detachment, retinal angiogenesis and choroidal neavasculzarzation (ChNV), posterior segment trauma, radiation retinopathy, epiretinal membrane or anterior ischemic optic neuropathy.

In some specific embodiments, the present disclosure provides a composition comprising the nanoparticles of the present disclosure.

Definition

All terms used in this article, including descriptive or technical terms, should be interpreted as having meanings that are obvious to a person of ordinary knowledge in the field. However, these terms may have different meanings depending on the intention of a person of ordinary knowledge in the field, previous cases, or the emergence of new technologies. In addition, the applicant may arbitrarily select some of the terms, in which case the meaning of the selected terms will be explained in detail in the description of this disclosure. Therefore, the terms in this article are defined based on the meaning of the terms and the overall description in the specification.

In this document, unless expressly and unambiguously limited to one referent, the terms "a", "an" or "the" include multiple referents. Unless the context clearly indicates otherwise, the term "or" can be used interchangeably with the term "and/or". The term "about" refers to an acceptable range or deviation of a value based on how it is measured or determined. In this document, the term "about" is understood to mean up to plus or minus 20% of a specific value, such as plus 1%, plus 5%, plus 10%, plus 15%, plus 20%, minus 1%, minus 5%, minus 10%, minus 15% or minus 20% of a specific value. In addition, when a component "includes" or "comprises" an element or step, unless there is a specific description to the contrary, the component may also include other components or other steps, rather than excluding other components or steps.

In this document, the numerical values or ranges listed as parameters are intended to include the numerical values and ranges between the listed numerical values or ranges. It should also be understood that the ranges in the present disclosure include lower limits and upper limits. For example, if a peptide has 12 to 50 amino acids, all individual numerical values such as 12, 13, 49, 50 and their sub-ranges such as 12 to 20, 20 to 30 and 30 to 50 are explicitly listed. All possible combinations of values between the lower limit and the upper limit are deemed to be explicitly indicated in the present disclosure.

The terms "self-assembled" and "self-assembly" refer to the phenomenon whereby the components of an object (e.g., the nanoparticles disclosed herein) organize themselves into a functional structure as a result of their own interactions, without external guidance. The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a warm-blooded animal that suffers from, is suspected of suffering from, is at risk of, will suffer from, or is being screened for angiogenesis or angiogenesis-related disorder, including those that actually suffer from or are suspected of suffering from the disease. Such terms include, but are not limited to, domestic animals, sports animals, primates, and humans. For example, mammals, preferably humans.

As used herein, the term "treat" or "treating" refers to actions taken to manage and care for a disease, disorder, or condition in an individual. The term is intended to include slowing the progression of the disease, disorder, or condition, reducing or alleviating symptoms and complications, and/or curing or eliminating the disease, disorder, or condition. The individual to be treated may be a mammal, such as a human.

The terms "reduce", "alleviate" or "inhibit" refer to a decrease in a parameter (e.g., angiogenesis) as measured by known standard means, wherein the parameter is reduced or at least reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more compared to an untreated individual.

The term "effective" means that the symptoms of a disease, disorder or condition are improved relative to an untreated individual. The amount of active compound used in the present disclosure that is effective for treating angiogenesis or angiogenesis-related diseases depends on the age, weight, general health, sex, diet, time of administration, drug interactions and severity of the individual's condition. The attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective" dose. If necessary or desired, treatment may involve multiple administrations of an effective amount of the nanoparticles or compositions of the present disclosure to the patient.

As used herein, the term "neovascularization" refers to the formation of new blood vessels. This process of creating new blood vessels can be triggered by environmental stimuli and mediated by angiogenesis and/or pathological tissue responses. For example, injured tissue may trigger a collection of pro-angiogenic factors that stimulate the release of proteases and ultimately lead to the formation and remodeling of new blood vessels. The term "angiogenesis" refers to the proliferation or growth of new blood vessels from existing blood vessels. Angiogenesis can be measured by measuring the total length of vascular segments per unit area, functional vascular density (total length of perfused vessels per unit area), or vascular volume density (total vascular volume per unit volume of tissue).

The term "treat" or any linguistic variation thereof refers to the administration of a therapeutically effective amount of the disclosed composition, which can effectively improve undesirable symptoms associated with a disease, prevent the manifestation of such disease before symptoms appear, slow the progression of the disease, slow the deterioration of symptoms, enhance the onset of remission, slow the irreversible damage caused by the progressive chronic stage of the disease, delay the onset of the progressive stage, reduce the severity of the disease or cure the disease, bring about rapid recovery, or prevent the occurrence of the disease or a combination of two or more of the above.

As used herein, the term "peptide" refers to a series of amino acid residues, usually linked to each other by peptide bonds between the α-amine and carbonyl groups of adjacent amino acids. In some embodiments, the peptides used in the present disclosure are 5 to 200 amino acids in length. In some embodiments, the peptides used in the present disclosure are 10 to 100 amino acids in length. In some embodiments, the peptides used in the present disclosure are 12 to 50 amino acids in length, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length.

In addition, the term "peptide" shall include a range of salts of amino acid residues, usually linked to each other by peptide bonds between the α-amine and carbonyl groups of adjacent amino acids. For example, inorganic ionic salts prepared from calcium, potassium, sodium, magnesium and their analogs; inorganic acid salts prepared from hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid and their analogs; trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid; Organic acid salts prepared from glutaric acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc.; sulfonic acid salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid and their analogs; amino acid salts prepared from glycine, arginine, lysine, etc.; amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc. and their analogs. Preferably, the salt is a pharmaceutically acceptable salt of a peptide.

The term "pharmaceutically acceptable excipient" used herein refers to a substance that facilitates the administration of an active agent to an individual and/or its absorption by an individual. In some specific aspects of the present disclosure, pharmaceutical excipients include but are not limited to fillers, binders, preservatives, polymers, solvents, antioxidants, disintegrants, suspending agents, wetting agents, lubricants, coatings, metals, sweeteners, flavoring agents, stabilizers or coloring agents. A person of ordinary skill in the art will understand that other pharmaceutical excipients may be used in the present disclosure.

The term "biocompatible" substance refers to a substance that does not generally cause adverse reactions when administered to an individual. Adverse reactions include, but are not limited to, significant inflammation and/or acute rejection of the substance by the individual's immune system, such as through T cell-mediated reactions. In some embodiments, the biocompatible substance is biodegradable. Non-limiting examples of biocompatible substances are biocompatible polymers (including biocompatible copolymers).

The application contains at least one color drawing. The Patent Office will provide copies of this patent application publication with color drawings upon request and the necessary fee.

By combining the attached figures and referring to the following description, it will be easier to understand the content of this disclosure.

Figure 1A shows a schematic diagram of the formation of self-assembled HA-peptide composite nanoparticles (HA-NPs).

FIG1B shows a schematic diagram showing the administration routes of positively charged HA-NPs (HA-NPs ⁺ ) and negatively charged HA-NPs (HA-NPs ⁻ ).

Figure 2 illustrates the morphology of HA-NPs ⁺ and HA-NPs ^- under transmission electron microscopy (TEM).

FIG3 shows the cumulative drug release rate of HA-NPs ⁺ , HA-NPs ⁻ , and pure gp91 ds-tat (gp) in a neutral (pH 7) environment of phosphate buffer solution (PBS).

Figure 4 shows the results of the cell survival test of human umbilical vein endothelial cells (HUVECs) treated with different concentrations of hyaluronic acid (HA), pure gp91 ds-tat (gp) and HA-NPs for one day.

Figures 5A to 5B illustrate the cellular uptake of free gp91 ds-tat (gp) and HA-NPs ⁺ in HUVECs. Figure 5A shows the cell counts by flow cytometry with FITC fluorescence labeling. Figure 5B shows a histogram of FITC intensity based on Figure 4.

Figures 6A to 6B depict the results of tube formation assays of HUVECs treated with hyaluronic acid (HA), pure gp91 ds-tat (gp), or HA-NPs ⁺ at 4, 16, and 24 hours after administration. Figure 6B shows a histogram of the number of branches based on Figure 5.

Figures 7A to 7B illustrate the drug retention rate of pure gp91 ds-tat (gp) or HA-NPs ⁺ in the mouse anterior eye over time. Figure 7A shows the fluorescence level in the mouse eye, where the minimum color scale is 9.69×10 ⁸ and the maximum color scale is 1.10×10 ¹⁰ . Figure 7B shows the quantitative results of the fluorescence intensity according to Figure 7A.

Figures 8A to 8B illustrate the treatment results of corneal neovascularization (CoNV) in chemically burned mice treated with PBS, hyaluronic acid (HA), gp91 ds-tat (gp) or HA-NPs ⁺ . Figure 8A shows the recovery progress of CoNV mouse eyes in each treatment group. Figure 8B shows a histogram of the vascular ratio in each treatment group according to Figure 7.

Figures 9A to 9B show the results of fluorescent frozen sections of mice treated with pure gp91 ds-tat (gp) and HA- ^NPs . Figure 9A shows an eye image in the choroid and retinal regions, in which TAMRA-labeled gp91 ds-tat is shown in red. Figure 9B shows a histogram of fluorescence intensity according to Figure 9A.

Figures 10A to 10B illustrate the effect of HA-NPs on the treatment of choroidal neovascularization (ChNV). Figure 10A shows fundus images of mice 7 days before laser-induced treatment (-7 days), on the day of laser-induced treatment (0 days), 7 days after laser-induced treatment (+7 days), and 14 days after laser-induced treatment (+14 days). Figure 10B shows a histogram of changes in angiogenesis area after PBS and HA-NPs ^- treatment. Normalized data based on day 0 are presented to describe fold changes.

The general feature of the present invention is a self-assembled nanoparticle, which contains a specific ratio of hyaluronic acid (HA) and a therapeutic peptide reagent and a composition thereof, which can be used to inhibit angiogenesis. The present invention also relates to a method for treating angiogenesis or angiogenesis-related diseases, comprising administering the above-mentioned nanoparticles or their compositions to an individual in need thereof. As described in more detail below, the combination of Nox2-targeted inhibitory peptide gp91 ds-tat and HA is identified as having the ability to inhibit ocular angiogenesis or angiogenesis, and the drug is retained in the eye for a longer time compared to the group treated with conventional pure gp91 or PBS.

Corneal neovascularization (CoNV)

The front 1/6 of the eyeball is the cornea, which covers the pupil, anterior chamber and iris. It is a transparent avascular structure. To maintain transparency, the cornea has no blood vessels. It mainly obtains nutrients through the vascular network at the edge of the cornea, followed by tears and aqueous humor. It obtains about 80% of oxygen directly from the air. Since the cornea is in direct contact with the air, it is prone to various infections and irritations. Once the cornea is irritated, the blood vessels of the vascular network at the edge of the cornea will proliferate forward. If no action is taken, it may lead to corneal disease, degeneration, and even blindness. There are many causes of CoNV, including but not limited to corneal immune response, microbial infection, corneal blister lesions, chemical damage (alkali damage and silver nitrate), congenital corneal lesions (Peter's deformity), vitamin A or other vitamins and amino acid deficiencies and metabolic disturbances, wearing soft/hard contact lenses, radial keratotomy, etc.

Hypoxia may also play a role in the pathogenesis of CoNV, as neovascularization is rarely observed in the eyes of individuals wearing gas-permeable lenses. Previous studies have shown that the main factors causing CoNV are infection, systemic disease, chemical burns, and aniridia. In summary, corneal neovascularization is one of the most serious ocular diseases currently.

Angiogenic pathways

Kim et al. (Blood. 123(5): p. 625-631, 2014) explained the angiogenic pathway. When NADPH oxidase is activated, vascular endothelial growth factor (VEGF) stimulates the production of reactive oxygen species (ROS). The increased ROS then participate in the phosphorylation of VEGFR2, which drives blood vessel proliferation (angiogenesis). NOX2, also known as gp91phox or CYBB, is the prototype of NADPH oxidase. Amphioxus NOX2 may be the origin of all mammalian NOX1/2/3. Gp91phox can severely inhibit reactive oxygen species to cause hypoxia. NADPH oxidase is the major source of peroxide production during hypoxia and has been shown to act on oxygen sensors activated by hypoxia in all cell types. During activation, the cytoplasmic subunits of NADPH oxidase (p47phox, p67phox, p40phox, Rac1, and Rac2) migrate to the membrane and dock with the membrane-bound subunits (gp91phox, p22phox).

Drugs for corneal neovascularization (CoNV)

The goal of CoNV treatment is to achieve anti-angiogenesis or vascular regression. Non-invasive treatments such as eye drops and oral medications have poor therapeutic effects. Current CoNV drugs such as bevacizumab, tocilizumab, and ranibizumab mainly target VEGF, because VEGF inhibition has shown significant effects in the treatment of CoNV. Today, drug treatments such as steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-VEGF agents, and cyclosporine have emerged. The first-line treatment of CoNV is steroids and nonsteroidal anti-inflammatory drugs. However, the risks of using steroids are that they may increase the chance of infection, glaucoma, cataracts, and recurrence of herpes simplex, while anti-inflammatory drugs may cause corneal ulceration and melting. A new treatment in recent years is anti-VEGF drugs (ranibizumab or bevacizumab), which have been shown to reduce CoNV. The vascular endothelial growth factor (VEGF) family and its receptor system have been shown to be the basic regulators of cellular signaling for angiogenesis. In addition, many studies have shown that VEGF plays an important role in angiogenesis and pathological angiogenesis associated with eye diseases. A potential treatment for CoNV is to inhibit VEGF activity by competing for the binding of VEGF with specific neutralizing anti-VEGF antibodies. However, anti-VEGF drugs have limitations in the treatment of CoNV. It is not a completely curative treatment and requires repeated treatment to maintain positive effects over time. Although bevacizumab or ranibizumab are anticancer drugs, they have been shown to be safe and effective in the short term, but long-term effects have not been documented and may cause corneal thinning, reduced epithelial healing, and epithelial erosion. Anti-VEGF therapy is currently still an experimental treatment. If the cornea is inflamed due to corneal neovascularization, inhibition of the enzyme can block CoNV by compromising corneal structural integrity. CoNV can also be suppressed by a combination of oral doxycycline tablets and topical corticosteroids.

New anti-angiogenic agent gp91 ds-tat

Gp91 ds-tat is a NADPH oxidase inhibitory peptide. It is a peptide that can block the binding of p47phox to NOX2 (gp91phox) and thus inhibit NAD(P)H activation. gp91 ds-tat prevents the binding of p47phox to Nox. Gp91 ds-tat contains a positively charged arginine residue, which gives the peptide a positive charge.

FERey et al. (Circulation research 89(5): p.408-414, 2001) demonstrated that gp91 can inhibit ROS formation and downregulate VEGF activity. In vitro experiments showed that when gp91 ds-tat was administered to angiotensin II-induced (Ang II) mice, O ₂ ^-values decreased. This showed that the inhibitor's ability to reduce vascular O ₂ ^- and blood pressure was significantly inhibited in the gp91 ds-tat group.

T.Usui et al. (Acta Physiologica 211(2): p.385-394, 2014) also demonstrated that gp91 ds-tat can inhibit ROS. They first studied whether brain-derived neurotrophic factor (BDNF) could induce ROS in human umbilical vein endothelial cells (HUVECs). Afterwards, HUVECs were pretreated with gp91 ds-tat before BDNF stimulation. The results showed that gp91 ds-tat could significantly inhibit BDNF-induced ROS generation, and its ROS signal intensity was lower than that of other groups.

Hiroki Hachisuka et al. (Journal of tissue engineering and regenerative Medicine 2(7): p.430-435, 2008) examined the effects of gp91 ds-tat on the growth of engineered tissue masses using a rat chamber model. They mixed Matrigel with gp91 ds-tat (100 Iza) and compared it with a control group to observe the formation of blood vessels. Although the total number of blood vessels per unit cellular area did not differ between the two groups, the majority of blood vessels in gp91 ds-tat-treated tissues had smaller columns compared with the control group (most blood in 10.3 in the control group and 1.7 in the gp91 ds-tat group; p<0.001). In vitro, gp91 ds-tat treatment reduced the proliferation and migration of cultured microvascular endothelial cells.

Therefore, it is believed that gp91 ds-tat may help inhibit corneal neovascularization (CoNV).

Nanomedicines for eye diseases

The complex drug delivery barriers in the eye reduce the bioavailability of many drugs in the eye and present poor therapeutic effects. Nanocarrier-mediated drugs can interact with the ocular mucosa, prolong the retention time, and increase the permeability of drugs on the cornea. In addition, it is necessary to reduce the toxicity and dosage of drugs. Therefore, nanoparticle drugs have become a potential technology for producing nanodrugs and applying them in ophthalmic treatment. Generally, the types of nanoparticles are divided into nanospheres, nanocapsules, polymer capsules, liposomes, fourth-generation dendrimers, ligand coupling and ferrofluid coupling ligands. The distribution of nanoparticles in the eye mainly depends on their size and surface properties. Nanoparticles with diameters ranging from 200 to 2000 nm can remain in ocular tissues for at least 2 months and can remain in precorneal tissues for a long time without being rapidly removed. Drugs can be encapsulated, bound or adsorbed in polymer nanoparticles.

Implementation example

Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed as limiting the scope of the present disclosure.

As shown in Figures 1A and 1B, the nanoparticles provided by the present invention are self-assembled composites of hyaluronic acid (HA) and therapeutic peptides. HA-peptide composite nanoparticles (HA-NPs) are colloids in which the charge of the particles can be positive or negative, depending on the ratio between HA and the peptide. Depending on their charge, HA-NPs can have different uses. As shown in Figure 1B, positively charged HA-NPs (HA-NPs ⁺ ) can be used to prepare eye drops, which can be applied to the anterior segment of the eye (e.g., the cornea or conjunctiva). Because the cornea and conjunctiva have negative surface charges, cationic colloidal HA-NPs can prolong retention and remain continuously on negatively charged ocular tissues. On the other hand, negatively charged HA-NPs (HA-NPs ^- ) can be administered to the posterior segment of the eye (e.g., retina, choroid, and sclera) via intravitreal injection without causing rejection events. Since the vitreous is composed of hyaluronic acid, HA-NPs located in the posterior segment of the eye can also avoid non-absorptive clearance mechanisms.

Example 1. Synthesis and characterization of self-assembled HA-NPs.

In the present disclosure, gp91 ds-tat (gp) was selected as an exemplary therapeutic peptide for preparing HA-NPs. Hyaluronic acid (HA) and gp solutions were prepared separately with specific concentrations of 1 to 5 mg/mL. To synthesize nanoparticles, 2 mL of deionized (DI) water was first added to a sample bottle and stirred at 990 rpm. HA and gp solutions were quickly added to the sample bottle at different addition ratios (HA: gp = 1: 0.5 to 2.5) to form nanoparticles with positive charge (HA-NPs ⁺ ) or negative charge (HA-NPs ^- ). These nanoparticles were stirred continuously for 10 minutes and then stored at 4°C for further use.

The particle size, zeta potential and polydispersity index (PdI) of HA-NPs ⁺ and HA-NPs ^- were measured at 25°C using a nanoparticle size analyzer (DLS) (Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK), and three measurements were performed for each group. To determine the peptide drug loading efficiency (EE) of the nanoparticles, HA-NPs ⁺ and HA-NPs ^- were centrifuged at 4000 rcf in a centrifugal filter tube for 20 minutes. Unloaded gp91 ds-tat was collected and quantified by protein assay reagent (Bio-Rad, USA). The peptide drug loading efficiency was calculated by the following formula:

The results are summarized in Tables 1 and 2 below. Table 1 shows that the charge of HA-NPs can be determined based on the ratio between HA and gp within the nanoparticles. An increase in gp in the ratio will result in more positively charged HA-NPs, while an increase in HA will result in more negatively charged HA-NPs.

Table 2 shows the average values of the properties of HA-NPs ⁺ and HA-NPs ^- . The average particle sizes of HA-NPs ⁺ and HA-NPs ^- measured by DLS were 268.4 ± 7.1 nm and 166.7 ± 1.0 nm, respectively. The zeta potentials of HA-NPs ⁺ and HA-NPs ^- were 17.9 ± 0.7 mV (positive) and -31.6 ± 0.6 mV (negative), respectively. The PdI values of all test groups were lower than 0.15, indicating that these samples were well-dispersed colloidal solutions. The peptide loading efficiency (EE) of HA-NPs ⁺ and HA-NPs ^- were around 98% and 76%, respectively, indicating good peptide loading.

Example 2. Morphological observation of HA-NPs by transmission electron microscopy (TEM)

The morphology of HA-NPs ⁺ and HA-NPs ^- was examined by transmission electron microscopy (TEM; HT-7700; Hitachi, Tokyo, Japan). Diluted HA-NPs were dropped on a nickel grid and then stained with 2% uranium acetate (UA) solution for 1 min. After drying, the samples were examined by TEM.

From the TEM images, round and spherical nanoparticles were observed in the HA-NPs ⁺ and HA-NPs ^- groups, and HA-NPs did not aggregate. The particle sizes were approximately 248 nm (HA-NPs ⁺ ) and 143 nm (HA-NPs ^- ) (Figure 2), which was roughly consistent with the particle size results obtained from DLS measurements.

Example 3. Evaluation of peptide/drug release rate

gp91 ds-tat (gp) was coupled to fluorescent isothiocyanate (FITC) for drug fluorescent labeling. FITC-labeled peptides were then used to prepare self-assembled HA-NPs ⁺ and HA-NPs ^- for peptide/drug release assessment. 1 mL of FITC-labeled HA-NPs ⁺ , HA-NPs ^- and pure gp solution were loaded into the dialysis membranes, respectively. Each dialysis membrane was then immersed in a total volume of 15 mL of PBS (pH 7) and incubated in a water bath with magnetic stirring at 37°C. At specific times (10 minutes, 30 minutes, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours), 1 mL of solution was collected from each group and then supplied with 1 mL of PBS. After all the collections were completed, the collected samples were tested for FITC concentration using a full-wavelength multi-function microplate analyzer (Varioskan Flash; Thermo Fisher Scientific, USA) with an excitation wavelength of 494 nm and an emission wavelength of 518 nm. The cumulative drug release rate of the FITC-labeled peptide was calculated and shown in Figure 3.

As shown in Figure 3, the peptide in the pure gp group was rapidly released within the first 24 hours (57.19% of the total amount) and reached a 100% release rate within 48 hours after the start of the evaluation. However, the release rate of HA-NPs was significantly slower than that of pure gp, as the cumulative release rates of the two were 8.9% (HA-NPs ⁺ ) and 7.2% (HA-NPs ^- ) within 24 hours after the start of the evaluation. After 72 hours of evaluation, HA-NPs ⁺ and HA-NPs ^- reached a release rate of 31.0% and 17.3%, respectively. The results showed that this revealed the sustained release state of peptide/drug when loaded in nanoparticles.

Example 4. Cell viability test

Human umbilical vein endothelial cells (HUVECs) were used for cell viability test. The cells were seeded in a 96-well plate (5×10 ³ cells/well) and cultured overnight. They were then cultured for one day with various concentrations of hyaluronic acid (HA), pure gp91 ds-tat (gp), HA-NPs ⁺ , and HA-NPs ^- . The cell viability of each treatment group was determined using a cell viability reagent (CCK-8). After culturing each group for 1 day, the culture medium was removed, and then 110 μL of culture medium containing CCK-8 was added to each group. After 3 hours of incubation, the reaction solution was tested at 450 nm using a full spectrum analyzer (EPOCH2; BioTek, USA). The results are shown as normalized data based on the control group (C) (Figure 4).

As shown in Figure 4, the cell viability of all treatment groups was higher than 70%, indicating that no biological toxicity was observed. Low concentrations (25 and 75 μg /mL) of HA-NPs did not affect cell viability. In addition, the charge of HA-NPs did not affect cell viability. It is worth noting that the treatment groups with HA-NPs concentrations higher than 100 μg/mL showed lower cell viability than the gp group. These results indicate that HA-NPs are non-toxic to cells.

Example 5. Cellular uptake of HA-NPs

FITC-labeled gp91 ds-tat was used to prepare HA-NPs ⁺ to track its fluorescence signal in flow cytometric assay. HUVECs were seeded in 24-well plates (2 x10 ⁵ cells/well) overnight. Pure gp91 ds-tat (gp) and HA-NPs ⁺ drug solution at a peptide concentration of 300 μg/mL were added to the cells. After 0.5 h and 2 h of incubation, cells were harvested and resuspended in PBS to form a single cell suspension. The cells were then assayed using a flow cytometer (Invitrogen Attune ^™ NxT Acoustic Focusing Cytometer; AFC2, Thermo, Singapore), and 1×10 ⁴ cells were counted and analyzed using AttuneNxT software (Invitrogen Attune ^™ NxT Acoustic Focusing Cytometer; AFC2, Thermo, Singapore).

As shown in Figures 5A and 5B, the HA-NPs ⁺ group showed significantly higher FITC intensity than the gp group at 0.5 hours of culture. Similar results were observed at 2 hours of culture, and the HA-NP ⁺ group obtained a higher fluorescence intensity compared to the gp group. This result shows that the HA-NPs provided by the present disclosure are easily taken up by cells and can deliver therapeutic peptides to cells.

Example 6. Column formation test

To understand the anti-angiogenic effects of HA-NPs and the therapeutic peptides therein, a tube formation test was performed to observe the formation of vascular networks. Matrigel ^TM hydrogel was used to provide HUVECs with an environment for tube formation. Matrigel was spread in the wells (150 μL) on a 48-well plate, and then these 48-well plates were incubated with Matrigel at 37°C for 30 minutes to solidify. HUVECs were prepared at a density of 1.5 x 10 ⁵ cells/well and given HA, gp, and HA-NPs ⁺ groups (100 μg/mL gp91 ds-tat peptide concentration), and then seeded in a 48-well plate. Each group was tested in triplicate, and cell images were taken at 4, 16, and 24 hours using an inverted fluorescence microscope (Leica DMi8, Germany). The number of branches was quantified by ImageJ software, and the branching growth of HUVECs represents the growth of blood vessels in vitro.

As shown in Figures 6A and 6B, the HA-NPs ⁺ group showed the lowest number of branches among all tested groups at all three time points. However, after 24 hours of treatment, HA-NPs ⁺ showed a significantly lower number of branches compared with the other groups. These results confirm that gp91 ds-tat nanoformulations embedded in HA (HA-NPs ⁺ ) can effectively inhibit the tube formation of HUVECs, and are more effective than the pure peptide and HA groups. The inhibition of tube formation also means that HA-NPs ⁺ can prevent angiogenesis in HUVECs.

Example 7. Drug retention of HA-NPs in the anterior segment of the eye

Animal experiments were performed on the anterior and posterior segments of the mouse eye using different administration routes, as shown in Figure 1B. For the anterior segment experiment, FITC-labeled gp91 ds-tat was formulated with HA to prepare HA-NPs ⁺ eye drops according to the above method. 10 μL of pure gp91 ds-tat (gp) and HA-NPs ⁺ eye drops with the same peptide concentration were applied to the eyes of C57BL/6 mice, and the fluorescence signals of the ocular surface were detected under anesthesia by IVIS® Lumina XRMS Small Animal Non-Invasive Live Imaging System (PerkinElmer; Waltham, MA). The amount of ocular fluorescence retention was examined at multiple time points (30 seconds, 5 minutes, 10 minutes, 30 minutes, 60 minutes).

Images of mouse eyes treated with different eye drops are shown in Figure 7A, and the quantitative data of the fluorescent signal (FITC) are summarized in Figure 7B. Three minutes after administration, the fluorescence intensity of the mouse eyes in the gp group was 61.8%±1.8%, and that in the HA-NPs ⁺ group was 73.7%±6.1%, indicating that HA-NPs ⁺ retained longer in the mouse eyes and similar results were observed after longer treatment times. Compared with pure peptide eye drops, HA-NPs ⁺ showed significant long-term retention in the anterior segment of the eye. The results show that HA-NPs can remain at the site of action for a long time, thus providing sustained drug release and reducing the frequency of repeated drug administration.

Example 8. The therapeutic effect of HA-NPs on corneal neovascularization (CoNV)

Male C57BL/6 mice (6 to 8 weeks old) were anesthetized by intraperitoneal injection of a mixture of Zoletil 50 ^® and Rompun ^® , and their eyes were topically anesthetized with 0.5% Alcaine ® (Alcon; Geneva, Switzerland). The corneas of mice were burned for 8 seconds using a 75% silver nitrate/25% potassium nitrate rod (1590; Grafco, Australia) to induce corneal neovascularization (CoNV). 10 μL of PBS, hyaluronic acid (HA), pure gp91 ds-tat (gp), and HA-NPs ⁺ were administered via eye drops every two days, and each test group (PBS, HA, gp, and HA-NP ⁺ ) was repeated 6 to 8 times. On days 0, 4, and 7 after administration, a slit lamp (Kowa, Japan) was used to observe the growth of blood vessels toward the center of the cornea and take photos. To evaluate the therapeutic effect of HA-NPs ⁺ on CoNV, the corneal blood vessel area of different treatments was quantified using ImageJ software and compared in Figure 8B.

The angiogenesis in the cornea is shown in Figure 8A, in which the PBS-treated eyes showed severe angiogenesis at 4 and 7 days after administration. In the HA, gp and HA-NPs ⁺ groups, especially in the HA-NPs ⁺ group, fewer corneal blood vessels were observed. Figure 8B also shows that HA-NPs ⁺ has the fastest and strongest anti-angiogenic effect on the wound, and exhibits the best effect in the treatment of CoNV in the form of eye drops. Combined with the results of the aforementioned Example 7, HA-NPs ⁺ eye drops can not only retain a longer drug exposure time on the ocular surface, but also provide an effective therapeutic effect of inhibiting angiogenesis for CoNV.

Example 9. Drug retention of HA-NPs in the posterior segment of the eye

To evaluate the drug retention of HA-NPs in the posterior segment of the eye, HA-NPs labeled with ((5-(6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA) quencher ^were intravitreally injected into mouse eyes. Mice were sacrificed 4 and 24 hours later, and the eyeballs were removed for cryosectioning. The sections were cut into 10 μm thickness using a cryo-microtome (CM 3505S, Leica, Germany) at -20 °C and then mounted on positively charged slides (Superfrost® Plus, Thermo). Cell nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI, 300 nM) and coverslips were applied. FAXS (Tissue Gnostics, Vienna, Austria) scans of frozen whole eye sections are shown in Figure 9A, where red dots represent the locations of TAMRA-labeled pure gp91 ds-tat(gp) or HA-NPs ^- particles.

In the gp group, strong signals were observed in the retinal and choroid regions 4 hours after administration (Figure 9B), indicating that the pure gp91 ds-tat peptide quickly moved from the vitreous to the retina and choroid. Then a significant decrease in fluorescence intensity was observed 24 hours after administration, indicating that the pure peptide could not be retained on the tissue. In contrast, HA- ^NPs- did not show strong fluorescence intensity within the first 4 hours, but the intensity increased significantly 24 hours after administration. The results showed that compared with pure peptides, HA-NPs administered by intravitreal injection did not cause a sudden release of the drug and could be highly retained in the posterior eye for a longer period of time.

Example 10. The therapeutic effect of HA-NPs on choroidal neovascularization (ChNV)

C57BL/6 mice were generally anesthetized as described above, and topical mydriasis (Mydrin®; Shiga, Japan) was performed before the experiment. The laser light was applied by Imagine Guide Laser System (Phoenix Research Laboratories, Tempe, AZ, USA), and the choroidal neovascularization (ChNV) formation was examined by fundus angiographic analysis (FFA). The mouse eyes were covered with 2% dry eye gel (OmniVision, SA, Neuhausen, Switzerland), and fundus images and fluorescein angiography images were taken using Phoenix MICRONh Lab (Phoenix Research Laboratories, Tempe, AZ, USA). Images of the posterior segment of the eye were initially taken with white light, and fluorescein angiography was performed by subcutaneously injecting 10% sodium fluorescein into the mice. The images taken from FFA to evaluate the changes in the angiogenesis area were then analyzed by ImageJ software. The healthy fundus contours of mice were recorded before laser-induced treatment of ChNV. The ChNV model was induced by laser on day 0, and then PBS/HA- ^NPs- were delivered intravitreally (IV) on day 1, and FFA was performed every week to examine angiogenesis to verify its efficacy. The results for 2 weeks are summarized in Figure 10A.

As shown in Figure 10A, the -7 day group (before laser-induced treatment) showed a healthy and normal fundus. Four laser spots were clearly observed after laser induction on day 0 (0 day group). After vitreous injection of HA-NPs ^- , the fundus laser-induced area was significantly smaller than that in the PBS group. Figure 10B shows the standard multiple change of neovascularization area compared with day 0. The PBS group showed an increase in neovascularization area at 7 and 14 days after treatment, while the HA-NPs group significantly reduced the level of neovascularization. The results showed that HA-NPs ^- effectively inhibited choroidal angiogenesis by reducing the area of neovascularization, indicating that HA-NPs ^- has great potential for application as an anti-angiogenic agent in the treatment of ChNV.

The present disclosure has been described with reference to its specific embodiments, and it should be understood that various modifications are based on the specific embodiments of the present disclosure without departing from the scope of the present disclosure. Therefore, the specific embodiments described herein are intended to cover modifications within the scope of the present disclosure, rather than to limit the present disclosure. Therefore, the broadest interpretation of the scope of the patent application should be given to cover all such modifications.

Claims (7)

A self-assembled nanoparticle comprising hyaluronic acid and a therapeutic peptide, wherein the nanoparticle is loaded with at least 70% w/w of a given amount of the therapeutic peptide, the particle size of the nanoparticle is 100 to 400 nm, the polydispersity index of the nanoparticle is 0.001 to 0.7, the Zeta potential of the nanoparticle is -40 mV to 40 mV, and the therapeutic peptide is a gp91 ds-tat peptide. The self-assembled nanoparticles as described in claim 1, wherein the therapeutic peptide has anti-angiogenic properties in vascular endothelial cells. A use of the self-assembled nanoparticles as described in claim 1, which is used to prepare a drug for inhibiting the formation or growth of blood vessels in an individual in need thereof, wherein the blood vessels are ocular blood vessels. The use as described in claim 3, wherein the nanoparticles are substantially positively charged and are administered to the anterior segment of the eye of the individual via eye drops, or the nanoparticles are substantially negatively charged and are administered to the posterior segment of the eye of the individual via vitreous injection. The use as described in claim 4, wherein the anterior segment of the eye includes the cornea or conjunctiva, the posterior segment of the eye includes the retina, choroid or sclera, and the therapeutic peptide inhibits the generation of reactive oxygen species (ROS), thereby inhibiting the expression of vascular endothelial growth factor (VEGF). The use as described in claim 3, wherein the individual suffers from ocular angiogenesis or angiogenesis-related disease. The use as described in claim 6, wherein the individual suffers from an ocular angiogenesis or angiogenesis-related disease, and the ocular angiogenesis-related disease is selected from degenerative macular degeneration (AMD), retinal artery or vein occlusion, retinal branch vein occlusion, retinopathy of prematurity (ROP), neovascular glaucoma, corneal angiogenesis, diabetic macular edema (DME), acute idiopathic macular degeneration, polypoidal choroidal vasculopathy, ischemic proliferative retinopathy, pigmentary retinitis (RP), cone cell dystrophy, Behcet’s disease, proliferative vitreoretinopathy (PVR), retinitis, uveitis, Leber’s hereditary optic neuropathy (Leber’s hereditary optic neuropathy), At least one of the following: retinal detachment, retinal pigment epithelial detachment, retinal angiogenesis and choroidal neovascularization (ChNV), posterior segment trauma, radiation retinopathy, epiretinal membrane and anterior ischemic optic neuropathy.

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